KEY POINTS:We developed assays to measure genetic variant effects on polycystin-1, the protein mutated in most autosomal dominant polycystic kidney disease. All tested pathogenic variants disrupted either polycystin-1 ciliary trafficking or channel function. Trafficking and channel function of some pathogenic variants was restored by low temperature culture to promote polycystin folding. BACKGROUND:Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of ADPKD, limited mechanistic understanding has hindered therapeutic development. Most ADPKD is caused by loss-of-function variants in polycystin-1 (PC1). METHODS:We developed assays that quantify the effect of nontruncating variants on PC1 ciliary localization, membrane trafficking, and polycystin channel function. RESULTS:We evaluated 29 nontruncating variants in PC1 and found that pathogenic variants disrupt two molecular phenotypes: ( 1 ) localization of PC1 at the primary cilium or ( 2 ) polycystin ion channel activity. Ciliary localization of a subset of polycystin variants was restored when cells were cultured at low temperature. A subset of variants with localization restored by low temperature formed functional channels. CONCLUSIONS:This study demonstrated that disruptions in polycystin ciliary trafficking and channel function are common causes of ADPKD. Defects in ciliary trafficking and channel function can be rescued for a subset of pathogenic variants, establishing a foundation for polycystin-targeted therapies in ADPKD.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [177, 1080]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [734]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [404, 690, 1163, 258]. The established, or potential, involvement of TRP channels in disease [1134] is reviewed in [452, 689], [692] and [468], together with a special edition of Biochemica et Biophysica Acta on the subject [689]. Additional disease related reviews, for pain [637], stroke [1143], sensation and inflammation [994], itch [130], and airway disease [313, 1058], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [810]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [1015, 693, 806]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [295]). TRPA1 activation of sensory neurons contribute to nociception [417, 895, 606]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [579, 60, 368, 581]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [26, 60]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [428, 515, 1089, 1088]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [429, 213]. The electron cryo-EM structure of TRPA1 [745] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [286, 783, 18, 4, 94, 450, 744, 70]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [770, 959, 1080]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [18] and [451]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [746, 18, 775, 825, 1129, 157, 730, 64, 158]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [350, 349]. Activation of TRPC channels by lipids is discussed by [70]. Important progress has been recently made in TRPC pharmacology [810, 623, 440, 102, 856, 192, 293]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [298, 71, 890, 1038, 1032, 154, 103, 565, 918, 412]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [230]. The structures of the apo and antagonist-bound states of TRPC1/TRPC4 heteromeric channels have been resolved by cryo-EM [1070]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1122, 727, 728, 1123, 543, 1176, 1117].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [350].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [277, 349, 746, 1159]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [401, 712]. TRPM3 (reviewed by [718]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [947]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [717, 946]. TRPM3 may contribute to the detection of noxious heat [1024]. TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells). The direct activators are calcium, adenosine diphosphate ribose (ADPR) [976] and cyclic ADPR (cADPR) [1126]. As for many ion channels, PI(4,5)P2 must also be present [1117]. Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [240]. Recent studies have reported structures of human (hs) TRPM2, which demonstrate two ADPR binding sites in hsTRPM2, one in the N-terminal MHR1/2 domain and the other in the C-terminal NUDT9-H domain. In addition, one Ca2+ binding site in the intracellular S2-S3 loop is revealed and proposed to mediate Ca2+ binding that induces conformational changes leading the ADPR-bound closed channel to open [390, 1034]. Meanwhile, a quadruple-residue motif (979FGQI982) was identified as the ion selectivity filter and a gate to control ion permeation in hsTRPM2 [1128]. TRPM2 is involved in warmth sensation [853], and contributes to several diseases [76]. TRPM2 interacts with extra synaptic NMDA receptors (NMDAR) and enhances NMDAR activity in ischemic stroke [1172]. Activation of TRPM2 in macrophages promotes atherosclerosis [1173, 1155]. Moreover, silica nanoparticles induce lung inflammation in mice via ROS/PARP/TRPM2 signaling-mediated lysosome impairment and autophagy dysfunction [1035]. Recent studies have designed various compounds for their potential to selectively inhibit the TRPM2 channel, including ACA derivatives A23, and 2,3-dihydroquinazolin-4(1H)-one derivatives [1145, 1147]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [1080]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [330]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [597]. Mutations are associated with conduction defects [407, 597, 884]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [999] and dendritic cell migration [52]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [541] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [517]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [247]. TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’) [173]. These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. PIP2 is required for TRPM6 and TRPM7 activation [815, 1085]. When either gene is deleted in mice, the result is embryonic lethality [416, 1073]. The C-terminal kinase region of TRPM6 and TRPM7 is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones [483, 484]. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [626]. The putative metal transporter proteins CNNM1-4 interact with TRPM7 and regulate TRPM7 channel activity [40, 471]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [63, 179, 225] reviewed by [1017, 566, 461, 653]. Direct chemical agonists include menthol and icilin[1094]. Besides, linalool can promote ERK phosphorylation in human dermal microvascular endothelial cells, down-regulate intracellular ATP levels, and activate TRPM8 [68]. Recent studies have found that TRPM8 has typical S4-S5 connectomes with clear selective filters and exowell rings [516], and have identified cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gated pathways [1119]. Moreover, the last 36 amino acids at the carboxyl terminal of TRPM8 are key protein sequences for TRPM8's temperature-sensitive function [195]. TRPM8 deficiency reduced the expression of S100A9 and increased the expression of HNF4α in the liver of mice, which reduced inflammation and fibrosis progression in mice with liver fibrosis, and helped to alleviate the symptoms of bile duct disease [560]. Channel deficiency also shortens the time of hypersensitivity reactions in migraine mouse models by promoting the recovery of normal sensitivity [12]. A cyclic peptide DeC‐1.2 was designed to inhibit ligand activation of TRPM8 but not cold activation, which can eliminate the side effects of cold dysalgesia in oxaliplatin-treated mice without changing body temperature [9]. Analysis of clinical data shows that TRPM8-specific blockers WS12 can reduce tumor growth in colorectal cancer xenografted mice by reducing transcription and activation of Wnt signaling regulators and β-catenin and its target oncogenes, such as C-Myc and Cyclin D1 [736]. TRPML (mucolipin) familyThe TRPML family [787, 1140, 780, 1092, 191] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [827]. TRPML2 and TRPML3 show increased channel activity in low luminal sodium and/or increased luminal pH, and are activated by similar small molecules [322, 147, 882]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [787, 694]). TRPP (polycystin) familyThe TRPP family (reviewed by [217, 215, 303, 1068, 377]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [1080]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [348]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [910]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [1001]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6. TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [767, 887, 927]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [849]. The pharmacology of TRPV1 channels is discussed in detail in [332] and [1022]. TRPV2 is probably not a thermosensor in man [740], but has recently been implicated in innate immunity [551]. Functional TRPV2 expression is described in placental trophoblast cells of mouse [205]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [127, 534]. TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [1064, 206, 655, 272]).TRPV6 is reported to play a key role in calcium transport in the mouse placenta [1063].

The Concise Guide to Pharmacology 2025/26 marks the seventh edition in this series of biennial publications in the British Journal of Pharmacology. Presented in landscape format, the guide provides a comparative overview of the pharmacology of drug target families. The concise nature of the Concise Guide refers to the style of presentation, being clear, accessible, and well-structured, rather than the scope of the content, which spans approximately 500 pages. The Concise Guide summarises the key pharmacological properties of around 1900 human drug targets, and nearly 7000 interactions, involving around 4400 ligands. While the content is a substantially condensed version of the more detailed information and links available at the www.guidetopharmacology.org website, the printed guide serves as a permanent, citable, point-in-time record, that remains stable despite ongoing updates to the online database. The full contents of this publication can be found at https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70231. The Concise Guides provide expert-curated recommendations of 'Gold Standard' selective pharmacological tools, available either commercially or as donations, which enable the identification of individual drug targets or families of drug targets. While the Concise Guide offers a more streamlined overview, more comprehensive information, including detailed pharmacological profiles and links to multiple online databases, is available through the Guide to Pharmacology website. The 2025/26 edition of the Concise Guide is based on material current as of mid-2025, and supersedes all previous editions, including the 2023/24 Guide, and earlier Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), and as such provides official IUPHAR classification and nomenclature for human drug targets, where applicable. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. Each section includes nomenclature guidance, concise summaries, information of the best available pharmacological tools, key references, and suggestions for further reading.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [177, 1080]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [734]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [404, 690, 1163, 258]. The established, or potential, involvement of TRP channels in disease [1134] is reviewed in [452, 689], [692] and [468], together with a special edition of Biochemica et Biophysica Acta on the subject [689]. Additional disease related reviews, for pain [637], stroke [1143], sensation and inflammation [994], itch [130], and airway disease [313, 1058], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [810]. Most TRP channels are regulated by phosphoinositides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [1015, 693, 806]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [295]). TRPA1 activation of sensory neurons contribute to nociception [417, 895, 606]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [579, 60, 368, 581]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [26, 60]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [428, 515, 1089, 1088]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [429, 213]. The electron cryo-EM structure of TRPA1 [745] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [286, 783, 18, 4, 94, 450, 744, 70]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [770, 959, 1080]). A comprehensive listing of G protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [18] and [451]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or components of multimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [746, 18, 775, 825, 1129, 157, 730, 64, 158]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for STIM-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [350, 349]. Activation of TRPC channels by lipids is discussed by [70]. Important progress has been recently made in TRPC pharmacology [810, 623, 440, 102, 856, 192, 293]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [298, 71, 890, 1038, 1032, 154, 103, 565, 918, 412]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [230]. The structures of the apo and antagonist-bound states of TRPC1/TRPC4 heteromeric channels have been resolved by cryo-EM [1070]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1122, 727, 728, 1123, 543, 1176, 1117].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [350].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [277, 349, 746, 1159]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [401, 712]. TRPM3 (reviewed by [718]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [947]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [717, 946]. TRPM3 may contribute to the detection of noxious heat [1024]. TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells). The direct activators are calcium, adenosine diphosphate ribose (ADPR) [976] and cyclic ADPR (cADPR) [1126]. As for many ion channels, PI(4,5)P2 must also be present [1117]. Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [240]. Recent studies have reported structures of human (hs) TRPM2, which demonstrate two ADPR binding sites in hsTRPM2, one in the N-terminal MHR1/2 domain and the other in the C-terminal NUDT9-H domain. In addition, one Ca2+ binding site in the intracellular S2-S3 loop is revealed and proposed to mediate Ca2+ binding that induces conformational changes leading the ADPR-bound closed channel to open [390, 1034]. Meanwhile, a quadruple-residue motif (979FGQI982) was identified as the ion selectivity filter and a gate to control ion permeation in hsTRPM2 [1128]. TRPM2 is involved in warmth sensation [853], and contributes to several diseases [76]. TRPM2 interacts with extra synaptic NMDA receptors (NMDAR) and enhances NMDAR activity in ischemic stroke [1172]. Activation of TRPM2 in macrophages promotes atherosclerosis [1173, 1155]. Moreover, silica nanoparticles induce lung inflammation in mice via ROS/PARP/TRPM2 signaling-mediated lysosome impairment and autophagy dysfunction [1035]. Recent studies have designed various compounds for their potential to selectively inhibit the TRPM2 channel, including ACA derivatives A23, and 2,3-dihydroquinazolin-4(1H)-one derivatives [1145, 1147]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [1080]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [330]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [597]. Mutations are associated with conduction defects [407, 597, 884]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [999] and dendritic cell migration [52]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [541] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [517]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [247]. TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’) [173]. These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. PIP2 is required for TRPM6 and TRPM7 activation [815, 1085]. When either gene is deleted in mice, the result is embryonic lethality [416, 1073]. The C-terminal kinase region of TRPM6 and TRPM7 is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones [483, 484]. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [626]. The putative metal transporter proteins CNNM1-4 interact with TRPM7 and regulate TRPM7 channel activity [40, 471]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [63, 179, 225] reviewed by [1017, 566, 461, 653]. Direct chemical agonists include menthol and icilin[1094]. Besides, linalool can promote ERK phosphorylation in human dermal microvascular endothelial cells, down-regulate intracellular ATP levels, and activate TRPM8 [68]. Recent studies have found that TRPM8 has typical S4-S5 connectomes with clear selective filters and exowell rings [516], and have identified cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gated pathways [1119]. Moreover, the last 36 amino acids at the carboxyl terminal of TRPM8 are key protein sequences for TRPM8's temperature-sensitive function [195]. TRPM8 deficiency reduced the expression of S100A9 and increased the expression of HNF4α in the liver of mice, which reduced inflammation and fibrosis progression in mice with liver fibrosis, and helped to alleviate the symptoms of bile duct disease [560]. Channel deficiency also shortens the time of hypersensitivity reactions in migraine mouse models by promoting the recovery of normal sensitivity [12]. A cyclic peptide DeC‐1.2 was designed to inhibit ligand activation of TRPM8 but not cold activation, which can eliminate the side effects of cold dysalgesia in oxaliplatin-treated mice without changing body temperature [9]. Analysis of clinical data shows that TRPM8-specific blockers WS12 can reduce tumor growth in colorectal cancer xenografted mice by reducing transcription and activation of Wnt signaling regulators and β-catenin and its target oncogenes, such as C-Myc and Cyclin D1 [736]. TRPML (mucolipin) familyThe TRPML family [787, 1140, 780, 1092, 191] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [827]. TRPML2 and TRPML3 show increased channel activity in low luminal sodium and/or increased luminal pH, and are activated by similar small molecules [322, 147, 882]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [787, 694]). TRPP (polycystin) familyThe TRPP family (reviewed by [217, 215, 303, 1068, 377]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [1080]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [348]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [910]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [1001]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6. TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxious heat (reviewed by [767, 887, 927]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant-negative manner when co-expressed with TRPV1 [849]. The pharmacology of TRPV1 channels is discussed in detail in [332] and [1022]. TRPV2 is probably not a thermosensor in man [740], but has recently been implicated in innate immunity [551]. Functional TRPV2 expression is described in placental trophoblast cells of mouse [205]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [127, 534]. TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [1064, 206, 655, 272]).TRPV6 is reported to play a key role in calcium transport in the mouse placenta [1063].

PC-1 and PC-2 form a heteromeric ion channel complex (hereafter called the Polycystin complex) that is abundantly expressed on primary cilia of renal epithelial cells. Mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). The Polycystin complex forms a non-selective cation channel, yet the spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood, partially due to technical limitations posed by the tiny ciliary compartment. Here, we employ our novel assays to functionally reconstitute the polycystin complex in the plasma membrane. Using whole-cell and ciliary patch-clamp recordings we identified a ciliary enriched oxysterol, 7β,27-DHC, as a critical component required for activation of the polycystin complex. We identified a novel oxysterol binding pocket in PC-2 using molecular docking simulation. We also identified two amino acids within the PC-2 oxysterol binding pocket, E208 and R581, to be critical for 7β,27-DHC dependent polycystin activation in both the plasma membrane and ciliary compartment. Further, we can show that the pharmacological and genetic inhibition of oxysterol synthesis by carbenoxolone (CNX) reduces channel activity in primary cilia. Our findings identified a unique second messenger that regulates the polycystin complex. We hypothesize that cilia-enriched lipids license the polycystin complex to be functional only in the ciliary organelle, thus providing novel insights into the spatial regulation of the polycystin complex. Our results also establish a framework to target the same allosteric regulatory site in the polycystin complex to identify activators of the polycystin channels as novel therapeutic strategies for ADPKD.

Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of this monogenic disorder, our limited mechanistic understanding of ADPKD has hindered therapeutic development. Here, we successfully developed bioassays that functionally classify missense variants in polycystin-1 (PC1). Strikingly, ADPKD pathogenic missense variants cluster into two major categories: 1) those that disrupt polycystin cell surface localization or 2) those that attenuate polycystin ion channel activity. We found that polycystin channels with defective surface localization could be rescued with a small molecule. We propose that small-molecule-based strategies to improve polycystin cell surface localization and channel function will be effective therapies for ADPKD patients.

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [176, 1072]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [730]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [401, 686, 1155, 256]. The established, or potential, involvement of TRP channels in disease [1126] is reviewed in [448, 685], [688] and [464], together with a special edition of Biochemica et Biophysica Acta on the subject [685]. Additional disease related reviews, for pain [633], stroke [1135], sensation and inflammation [988], itch [130], and airway disease [310, 1051], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [805]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [1009, 689, 801]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [293]). TRPA1 activation of sensory neurons contribute to nociception [414, 890, 602]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [575, 60, 365, 577]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [26, 60]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [424, 511, 1081, 1080]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [425, 212]. The electron cryo-EM structure of TRPA1 [740] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [284, 778, 18, 4, 94, 446, 739, 70]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [765, 953, 1072]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [18] and [447]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [741, 18, 770, 820, 1121, 157, 726, 64, 158]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [347, 346]. Activation of TRPC channels by lipids is discussed by [70]. Important progress has been recently made in TRPC pharmacology [805, 619, 436, 102, 851, 191, 291]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [295, 71, 885, 1031, 1025, 154, 103, 561, 913, 409]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [229]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1114, 723, 724, 1115, 539, 1168, 1109].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [347].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [275, 346, 741, 1151]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [398, 708]. TRPM3 (reviewed by [714]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [941]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [713, 940]. TRPM3 may contribute to the detection of noxious heat [1017]. TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells). The direct activators are calcium, adenosine diphosphate ribose (ADPR) [970] and cyclic ADPR (cADPR) [1118]. As for many ion channels, PI(4,5)P2 must also be present [1109]. Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [239]. Recent studies have reported structures of human (hs) TRPM2, which demonstrate two ADPR binding sites in hsTRPM2, one in the N-terminal MHR1/2 domain and the other in the C-terminal NUDT9-H domain. In addition, one Ca2+ binding site in the intracellular S2-S3 loop is revealed and proposed to mediate Ca2+ binding that induces conformational changes leading the ADPR-bound closed channel to open [387, 1027]. Meanwhile, a quadruple-residue motif (979FGQI982) was identified as the ion selectivity filter and a gate to control ion permeation in hsTRPM2 [1120]. TRPM2 is involved in warmth sensation [848], and contributes to several diseases [76]. TRPM2 interacts with extra synaptic NMDA receptors (NMDAR) and enhances NMDAR activity in ischemic stroke [1164]. Activation of TRPM2 in macrophages promotes atherosclerosis [1165, 1147]. Moreover, silica nanoparticles induce lung inflammation in mice via ROS/PARP/TRPM2 signaling-mediated lysosome impairment and autophagy dysfunction [1028]. Recent studies have designed various compounds for their potential to selectively inhibit the TRPM2 channel, including ACA derivatives A23, and 2,3-dihydroquinazolin-4(1H)-one derivatives [1137, 1139]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [1072]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [327]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [593]. Mutations are associated with conduction defects [404, 593, 879]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [993] and dendritic cell migration [52]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [537] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [513]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [246]. TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’) [172]. These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. PIP2 is required for TRPM6 and TRPM7 activation [810, 1077]. When either gene is deleted in mice, the result is embryonic lethality [413, 1065]. The C-terminal kinase region of TRPM6 and TRPM7 is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones [479, 480]. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [622]. The putative metal transporter proteins CNNM1-4 interact with TRPM7 and regulate TRPM7 channel activity [40, 467]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [63, 178, 224] reviewed by [1011, 562, 457, 649]. Direct chemical agonists include menthol and icilin[1086]. Besides, linalool can promote ERK phosphorylation in human dermal microvascular endothelial cells, down-regulate intracellular ATP levels, and activate TRPM8 [68]. Recent studies have found that TRPM8 has typical S4-S5 connectomes with clear selective filters and exowell rings [512], and have identified cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gated pathways [1111]. Moreover, the last 36 amino acids at the carboxyl terminal of TRPM8 are key protein sequences for TRPM8's temperature-sensitive function [194]. TRPM8 deficiency reduced the expression of S100A9 and increased the expression of HNF4α in the liver of mice, which reduced inflammation and fibrosis progression in mice with liver fibrosis, and helped to alleviate the symptoms of bile duct disease [556]. Channel deficiency also shortens the time of hypersensitivity reactions in migraine mouse models by promoting the recovery of normal sensitivity [12]. A cyclic peptide DeC‐1.2 was designed to inhibit ligand activation of TRPM8 but not cold activation, which can eliminate the side effects of cold dysalgesia in oxaliplatin-treated mice without changing body temperature [9]. Analysis of clinical data shows that TRPM8-specific blockers WS12 can reduce tumor growth in colorectal cancer xenografted mice by reducing transcription and activation of Wnt signaling regulators and β-catenin and its target oncogenes, such as C-Myc and Cyclin D1 [732]. TRPML (mucolipin) familyThe TRPML family [782, 1132, 775, 1084, 190] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [822]. TRPML2 and TRPML3 show increased channel activity in low luminal sodium and/or increased luminal pH, and are activated by similar small molecules [319, 147, 877]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [782, 690]). TRPP (polycystin) familyThe TRPP family (reviewed by [216, 214, 300, 1061, 374]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [1072]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [345]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [905]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [995]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6. TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [762, 882, 922]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [844]. The pharmacology of TRPV1 channels is discussed in detail in [329] and [1015]. TRPV2 is probably not a thermosensor in man [736], but has recently been implicated in innate immunity [547]. Functional TRPV2 expression is described in placental trophoblast cells of mouse [204]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [127, 530]. TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [1057, 205, 651, 270]).TRPV6 is reported to play a key role in calcium transport in the mouse placenta [1056].

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The mechanism by which cells control the size of organelles is a fundamental question in cell biology. Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple structure makes them ideal for investigating the mechanisms of size regulation. Flagella are constructed and maintained via an active transport process called intraflagellar transport (IFT). Since no protein synthesis occurs in the flagellum, new materials must be injected into the flagellum by a process called IFT injection.

Cilia or eukaryotic flagella are microtubule-based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavorable to diffusive transport of most components required for their assembly and maintenance. Instead, a system of intraflagellar transport (IFT) trains moves cargo rapidly up and down the cilium (Figure 1A).1-3 Anterograde IFT, from the cell body to the ciliary tip, is driven by kinesin-II motors, whereas retrograde IFT is powered by cytoplasmic dynein-1b motors.4 Both motors are associated with long chains of IFT protein complexes, known as IFT trains, and their cargoes.5-8 The conversion from anterograde to retrograde motility at the ciliary tip involves (1) the dissoci-ation of kinesin motors from trains,9 (2) a fundamental restructuring of the train from the anterograde to the retrograde architecture,8,10,11 (3) the unloading and reloading of cargo,2 and (4) the activation of the dynein motors.8,12 A prominent hypothesis is that there is dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate these processes.4,13 However, the mechanisms of IFT turnaround have remained elusive. In this study, we use mechanical and chemical methods to block IFT at intermediate positions along the cilia of the green algae Chlamydomonas reinhardtii, in normal and calcium-depleted conditions. We show that IFT turnaround, kinesin dissociation, and dynein-1b activation can consistently be induced at arbitrary distances from the ciliary tip, with no stationary tip machinery being required. Instead, we demonstrate that the anterograde-to-retrograde conversion is a calcium-independent intrinsic ability of IFT.

The TRP superfamily of channels (nomenclature as agreed by NC-IUPHAR [159, 999]), whose founder member is the Drosophila Trp channel, exists in mammals as six families; TRPC, TRPM, TRPV, TRPA, TRPP and TRPML based on amino acid homologies. TRP subunits contain six putative TM domains and assemble as homo- or hetero-tetramers to form cation selective channels with diverse modes of activation and varied permeation properties (reviewed by [679]). Established, or potential, physiological functions of the individual members of the TRP families are discussed in detail in the recommended reviews and in a number of books [371, 635, 1066, 236]. The established, or potential, involvement of TRP channels in disease is reviewed in [412, 634] and [637], together with a special edition of Biochemica et Biophysica Acta on the subject [634]. Additional disease related reviews, for pain [585], stroke [1052], sensation and inflammation [921], itch [117], and airway disease [284, 979], are available. The pharmacology of most TRP channels has been advanced in recent years. Broad spectrum agents are listed in the tables along with more selective, or recently recognised, ligands that are flagged by the inclusion of a primary reference. See Rubaiy (2019) for a review of pharmacological tools for TRPC1/C4/C5 channels [751]. Most TRP channels are regulated by phosphoinostides such as PtIns(4,5)P2 although the effects reported are often complex, occasionally contradictory, and likely to be dependent upon experimental conditions, such as intracellular ATP levels (reviewed by [941, 638, 747]). Such regulation is generally not included in the tables.When thermosensitivity is mentioned, it refers specifically to a high Q10 of gating, often in the range of 10-30, but does not necessarily imply that the channel's function is to act as a 'hot' or 'cold' sensor. In general, the search for TRP activators has led to many claims for temperature sensing, mechanosensation, and lipid sensing. All proteins are of course sensitive to energies of binding, mechanical force, and temperature, but the issue is whether the proposed input is within a physiologically relevant range resulting in a response. TRPA (ankyrin) familyTRPA1 is the sole mammalian member of this group (reviewed by [268]). TRPA1 activation of sensory neurons contribute to nociception [382, 831, 555]. Pungent chemicals such as mustard oil (AITC), allicin, and cinnamaldehyde activate TRPA1 by modification of free thiol groups of cysteine side chains, especially those located in its amino terminus [529, 51, 336, 531]. Alkenals with α, β-unsaturated bonds, such as propenal (acrolein), butenal (crotylaldehyde), and 2-pentenal can react with free thiols via Michael addition and can activate TRPA1. However, potency appears to weaken as carbon chain length increases [23, 51]. Covalent modification leads to sustained activation of TRPA1. Chemicals including carvacrol, menthol, and local anesthetics reversibly activate TRPA1 by non-covalent binding [391, 470, 1007, 1006]. TRPA1 is not mechanosensitive under physiological conditions, but can be activated by cold temperatures [392, 193]. The electron cryo-EM structure of TRPA1 [688] indicates that it is a 6-TM homotetramer. Each subunit of the channel contains two short ‘pore helices’ pointing into the ion selectivity filter, which is big enough to allow permeation of partially hydrated Ca2+ ions. TRPC (canonical) familyMembers of the TRPC subfamily (reviewed by [261, 726, 15, 4, 84, 410, 687, 60]) fall into the subgroups outlined below. TRPC2 is a pseudogene in humans. It is generally accepted that all TRPC channels are activated downstream of Gq/11-coupled receptors, or receptor tyrosine kinases (reviewed by [713, 889, 999]). A comprehensive listing of G-protein coupled receptors that activate TRPC channels is given in [4]. Hetero-oligomeric complexes of TRPC channels and their association with proteins to form signalling complexes are detailed in [15] and [411]. TRPC channels have frequently been proposed to act as store-operated channels (SOCs) (or compenents of mulimeric complexes that form SOCs), activated by depletion of intracellular calcium stores (reviewed by [689, 15, 718, 765, 1039, 141, 675, 55, 142]). However, the weight of the evidence is that they are not directly gated by conventional store-operated mechanisms, as established for Stim-gated Orai channels. TRPC channels are not mechanically gated in physiologically relevant ranges of force. All members of the TRPC family are blocked by 2-APB and SKF96365 [319, 318]. Activation of TRPC channels by lipids is discussed by [60]. Important progress has been recently made in TRPC pharmacology [751, 571, 400, 92]. TRPC channels regulate a variety of physiological functions and are implicated in many human diseases [270, 61, 827, 960]. TRPC1/C4/C5 subgroup TRPC1 alone may not form a functional ion channel [210]. TRPC4/C5 may be distinguished from other TRP channels by their potentiation by micromolar concentrations of La3+. TRPC2 is a pseudogene in humans, but in other mammals appears to be an ion channel localized to microvilli of the vomeronasal organ. It is required for normal sexual behavior in response to pheromones in mice. It may also function in the main olfactory epithelia in mice [1036, 672, 673, 1037, 496, 1077, 1032].TRPC3/C6/C7 subgroup All members are activated by diacylglycerol independent of protein kinase C stimulation [319].TRPM (melastatin) familyMembers of the TRPM subfamily (reviewed by [252, 318, 689, 1064]) fall into the five subgroups outlined below. TRPM1/M3 subgroupIn darkness, glutamate released by the photoreceptors and ON-bipolar cells binds to the metabotropic glutamate receptor 6 , leading to activation of Go . This results in the closure of TRPM1. When the photoreceptors are stimulated by light, glutamate release is reduced, and TRPM1 channels are more active, resulting in cell membrane depolarization. Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB), whose patients lack rod function. TRPM1 is also found melanocytes. Isoforms of TRPM1 may present in melanocytes, melanoma, brain, and retina. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures [368, 657]. TRPM3 (reviewed by [663]) exists as multiple splice variants which differ significantly in their biophysical properties. TRPM3 is expressed in somatosensory neurons and may be important in development of heat hyperalgesia during inflammation (see review [878]). TRPM3 is frequently coexpressed with TRPA1 and TRPV1 in these neurons. TRPM3 is expressed in pancreatic beta cells as well as brain, pituitary gland, eye, kidney, and adipose tissue [662, 877]. TRPM3 may contribute to the detection of noxious heat [949].TRPM2TRPM2 is activated under conditions of oxidative stress (respiratory burst of phagocytic cells) and ischemic conditions. However, the direct activators are ADPR(P) and calcium. As for many ion channels, PIP2 must also be present (reviewed by [1020]). Numerous splice variants of TRPM2 exist which differ in their activation mechanisms [219]. The C-terminal domain contains a TRP motif, a coiled-coil region, and an enzymatic NUDT9 homologous domain. TRPM2 appears not to be activated by NAD, NAAD, or NAADP, but is directly activated by ADPRP (adenosine-5'-O-disphosphoribose phosphate) [902]. TRPM2 is involved in warmth sensation [789], and contributes to neurological diseases [66]. Recent study shows that 2'-deoxy-ADPR is an endogenous TRPM2 superagonist [253]. TRPM4/5 subgroupTRPM4 and TRPM5 have the distinction within all TRP channels of being impermeable to Ca2+ [999]. A splice variant of TRPM4 (i.e.TRPM4b) and TRPM5 are molecular candidates for endogenous calcium-activated cation (CAN) channels [301]. TRPM4 is active in the late phase of repolarization of the cardiac ventricular action potential. TRPM4 deletion or knockout enhances beta adrenergic-mediated inotropy [546]. Mutations are associated with conduction defects [374, 546, 821]. TRPM4 has been shown to be an important regulator of Ca2+ entry in to mast cells [926] and dendritic cell migration [43]. TRPM5 in taste receptor cells of the tongue appears essential for the transduction of sweet, amino acid and bitter stimuli [494] TRPM5 contributes to the slow afterdepolarization of layer 5 neurons in mouse prefrontal cortex [471]. Both TRPM4 and TRPM5 are required transduction of taste stimuli [226].TRPM6/7 subgroupTRPM6 and 7 combine channel and enzymatic activities (‘chanzymes’). These channels have the unusual property of permeation by divalent (Ca2+, Mg2+, Zn2+) and monovalent cations, high single channel conductances, but overall extremely small inward conductance when expressed to the plasma membrane. They are inhibited by internal Mg2+ at ~0.6 mM, around the free level of Mg2+ in cells. Whether they contribute to Mg2+ homeostasis is a contentious issue. When either gene is deleted in mice, the result is embryonic lethality. The C-terminal kinase region is cleaved under unknown stimuli, and the kinase phosphorylates nuclear histones. TRPM7 is responsible for oxidant- induced Zn2+ release from intracellular vesicles [3] and contributes to intestinal mineral absorption essential for postnatal survival [574]. TRPM8Is a channel activated by cooling and pharmacological agents evoking a ‘cool’ sensation and participates in the thermosensation of cold temperatures [54, 161, 205] reviewed by [943, 516, 420, 599]. TRPML (mucolipin) familyThe TRPML family [729, 1049, 723, 1010, 173] consists of three mammalian members (TRPML1-3). TRPML channels are probably restricted to intracellular vesicles and mutations in the gene (MCOLN1) encoding TRPML1 (mucolipin-1) cause the neurodegenerative disorder mucolipidosis type IV (MLIV) in man. TRPML1 is a cation selective ion channel that is important for sorting/transport of endosomes in the late endocytotic pathway and specifically, fission from late endosome-lysosome hybrid vesicles and lysosomal exocytosis [766]. TRPML2 and TRPML3 show increased channel activity in low extracellular sodium and are activated by similar small molecules [293]. A naturally occurring gain of function mutation in TRPML3 (i.e. A419P) results in the varitint waddler (Va) mouse phenotype (reviewed by [729, 639]). TRPP (polycystin) familyThe TRPP family (reviewed by [197, 195, 275, 988, 345]) or PKD2 family is comprised of PKD2 (PC2), PKD2L1 (PC2L1), PKD2L2 (PC2L2), which have been renamed TRPP1, TRPP2 and TRPP3, respectively [999]. It should also be noted that the nomenclature of PC2 was TRPP2 in old literature. However, PC2 has been uniformed to be called TRPP2 [317]. PKD2 family channels are clearly distinct from the PKD1 family, whose function is unknown. PKD1 and PKD2 form a hetero-oligomeric complex with a 1:3 ratio. [845]. Although still being sorted out, TRPP family members appear to be 6TM spanning nonselective cation channels. TRPV (vanilloid) familyMembers of the TRPV family (reviewed by [928]) can broadly be divided into the non-selective cation channels, TRPV1-4 and the more calcium selective channels TRPV5 and TRPV6.TRPV1-V4 subfamilyTRPV1 is involved in the development of thermal hyperalgesia following inflammation and may contribute to the detection of noxius heat (reviewed by [710, 824, 860]). Numerous splice variants of TRPV1 have been described, some of which modulate the activity of TRPV1, or act in a dominant negative manner when co-expressed with TRPV1 [787]. The pharmacology of TRPV1 channels is discussed in detail in [303] and [947]. TRPV2 is probably not a thermosensor in man [684], but has recently been implicated in innate immunity [503]. TRPV3 and TRPV4 are both thermosensitive. There are claims that TRPV4 is also mechanosensitive, but this has not been established to be within a physiological range in a native environment [114, 488].TRPV5/V6 subfamily TRPV5 and TRPV6 are highly expressed in placenta, bone, and kidney. Under physiological conditions, TRPV5 and TRPV6 are calcium selective channels involved in the absorption and reabsorption of calcium across intestinal and kidney tubule epithelia (reviewed by [984, 185, 601, 248]).

PC-1 and PC-2 form an ion channel complex called the polycystin complex, which predominantly localizes to a small hair-like organelle called the primary cilium. The polycystin complex permeates cations, K+, Na+, and Ca2+, and has an unusual 1:3 stoichiometry that combines one PC-1 subunit with three PC-2 subunits. However, the small size and shape of primary cilia impose technical challenges to study the polycystin complex in its native environment. In this paper, we describe the methodology to directly record ion channel activity in primary cilia. This method will allow a detailed functional characterization of how mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), essential to develop novel therapeutics for this ciliopathy.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point‐in‐time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid‐2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC‐IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Cilia and flagella are microtubule doublet based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavourable to diffusive transport for their assembly and maintenance. Instead, a highly dynamic system of intraflagellar transport (IFT) trains moves rapidly up and down the cilium. However, the mechanism of how these trains turn around upon reaching the ciliary tip has remained elusive. It has been hypothesized that there exists a dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate this conversion. In this work, we use physical and chemical methods to manipulate IFT in the cilia of the unicellular green alga Chlamydomonas reinhardtii to show that no such stationary tip-machinery is required for IFT turnaround. Instead, we demonstrate that the conversion from anterograde to retrograde IFT trains is a calcium independent intrinsic ability of the IFT system.

The left-right organizer (LRO) in the mouse consists of pit cells within the depression, located at the end of the developing notochord, also known as the embryonic node and crown cells lining the outer periphery of the node. Cilia on pit cells are posteriorly tilted, rotate clockwise and generate leftward fluid flow. Primary cilia on crown cells are required to interpret the directionality of fluid movement and initiate flow-dependent gene transcription. Crown cells express PC1-L1 and PC2, which may form a heteromeric polycystin channel complex on primary cilia. It is still only poorly understood how fluid flow activates the ciliary polycystin complex. Besides polycystin channels voltage gated channels like HCN4 and KCNQ1 have been implicated in establishing asymmetry. How this electrical network of ion channels initiates left-sided signalling cascades and differential gene expression is currently only poorly defined.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease. eLife digest On the surface of most animal and other eukaryotic cells are small rod-like protrusions known as primary cilia. Each cilium is encased by a specialized membrane which is enriched in protein complexes that help the cell sense its local environment. Some of these complexes help transport ions in out of the cell, while others act as receptors that receive chemical signals called ligands. A unique ion channel known as the polycystin complex is able to perform both of these roles as it contains a receptor called PC-1 in addition to an ion channel called PC-2. Various mutations in the genes that code for PC-1 and PC-2 can result in autosomal dominant polycystic kidney disease (ADPKD), which is the most common monogenetic disease in humans. However, due to the small size of primary cilia – which are less than a thousandth of a millimeter thick – little is known about how polycystin complexes are regulated and how mutations lead to ADPKD. To overcome this barrier, Ha et al. modified kidney cells grown in the lab so that PC-1 and PC-2 form a working channel in the plasma membrane which surrounds the entire cell. As the body of a cell is around 10,000 times bigger than the cilium, this allowed the movement of ions across the polycystin complex to be studied using conventional techniques. Experiments using this newly developed assay revealed that a region at one of the ends of the PC-1 protein, named the C-type lectin domain, is essential for stimulating polycystin complexes. Ha et al. found that this domain of PC-1 is able to cut itself from the protein complex. Further experiments showed that when fragments of PC-1, which contain the C-type lectin domain, are no longer bound to the membrane, they can activate the polycystin channels in cilia as well as the plasma membrane. This suggests that this region of PC-1 may also act as a secreted ligand that can activate other polycystin channels. Some of the genetic mutations that cause ADPKD likely disrupt the activity of the polycystin complex and reduce its ability to transport ions across the cilia membrane. Therefore, the cell assay created in this study could be used to screen for small molecules that can restore the activity of these ion channels in patients with ADPKD. Introduction The most common monogenetic disease in humans is ADPKD; a major nephropathy characterized by renal cysts that leads to urinary tract infection, hypertension, aneurysm, and ultimately end-stage renal disease (ESRD) (Kathem et al., 2014; Harris and Torres, 2009). Mutations in one of two ciliary proteins, PC-1 (encoded by PKD1) and PC-2 (also known as TRPP1 and encoded by PKD2), account for 85% and 15% of ADPKD-causing mutations, respectively (Harris and Torres, 2009; Cornec-Le Gall et al., 2014). PC-1 is an 11 transmembrane (TM) protein of 4303 amino acids with a long extracellular N-terminus that contains multiple cell adhesion domains (Yoder et al., 2002; Hughes et al., 1995). PC-1 and its family members (PC-1L1, PC-1L2, and PC-1L3) share the same 11-TM topology but vary in the size and adhesion domain composition of their N-termini (Ishimaru et al., 2006). A unique feature of PC-1 family members, shared only by adhesion G protein-coupled receptors (GPCRs), is the GPCR autoproteolysis inducing (GAIN) domain. This domain catalyzes peptide bond hydrolysis at a GPCR proteolysis site (GPS) that is positioned proximal to the first TM helix (TM1) (Araç et al., 2012; Prömel et al., 2013; Trudel et al., 2016). Autoproteolysis generates two fragments: an N-terminal fragment (NTF) and a multi-TM core (Qian et al., 2002). The NTF contains multiple domains, including leucine-rich repeats, a C-type lectin (CTL) domain, immunoglobulin (Ig)-like polycystic kidney disease (PKD) repeat domains, a single low-density lipoprotein (LDL) receptor motif, and the receptor for egg jelly (REJ) domain (Hughes et al., 1995; Babich et al., 2004; Moy et al., 1996). The function of the multi-TM core remains poorly defined. Autoproteolysis occurs early in the ER secretory pathway such that the N- terminal fragment remains non-covalently tethered (Lin et al., 2004; Wei et al., 2007). While mutations within the GPS site of PC-1 result in renal cyst formation, the full functional consequences of autoproteolysis are poorly understood (Qian et al., 2002; Yu et al., 2007). Recent studies suggest that PC-1 and PC-2 form a 1:3 heteromer (Su et al., 2018; Yu et al., 2009). Indeed, the cryo-EM structure of a PC-1/PC-2 heteromer reveals that the 11 transmembrane helices of PC-1 are further divided into two major domains: a peripheral TM1-TM5 complex and a core TM6-TM11 complex that interdigitates with three PC-2 subunits to form a TRP-like ion channel (Su et al., 2018). In addition to this PC-1/PC-2 assembly, PC-2 can form homomeric channels, following a classic homotetramer of TRP channels (Shen et al., 2016; Grieben et al., 2017; Wilkes et al., 2017). Attempts to exogenously express and record from PC-1 and/or PC-2 in whole cell recordings of mammalian cells (Hanaoka et al., 2000; Delmas et al., 2004; Cai et al., 2004) and reconstituted lipid bilayers González-Perrett et al., 2001 have yielded divergent results, likely due to subcellular enrichment of PC-1 and PC-2 (for a detailed discussion see Liu et al., 2018; Kleene and Kleene, 2017). In addition, despite recent advances in measuring ion channel activity in small subcellular organelles such as the cilium (Kleene and Kleene, 2012; DeCaen et al., 2013; Dong et al., 2008), it remains technically challenging to study ion channel activity in primary cilia due to their small size (10−15 L, approximately the size of a bacterium). This technical hurdle has hindered investigations of the physiological regulation of polycystins and how they lead to renal disease when mutated. The unknown role of PC-1 within the channel complex, in particular, has hindered progress in the field. Some studies suggest that PC-1 acts as a dominant-negative subunit of the heteromeric channel (Su et al., 2018), whereas others suggest that it is a pore-forming subunit that fine-tunes ion selectivity (Wang et al., 2019). Moreover, PC-1 has been shown to be dispensable for polycystin currents in ciliary membranes (Liu et al., 2018). Here we develop cellular assays that allow us to probe the function of polycystin complexes in the plasma membrane and compare them to polycystin channels in their native primary cilia. Our heterologous system recapitulates the biophysical properties of cilia-localized polycystin channels and demonstrates how distinct PC-1 family members contribute to ion permeation. Importantly, we reveal that the N-terminal domain of PC-1 functions as a soluble ligand and is an indispensable activator of the polycystin complex, providing further evidence for chemosensory regulation of the polycystin complex (Delling et al., 2016). Results PC-1 and PC-2 form functional channels in the plasma membrane Because robust assays to investigate the heteromeric polycystin complex are lacking, we developed an assay system to determine the contribution of PC-1 family members to polycystin function. We were able to target PC-1 to the plasma membrane of HEK293 cells (Figure 1B) and mIMCD3 cells (Figure 1—figure supplement 1E) by replacing its endogenous signal peptide with a strong Ig κ-chain secretion sequence followed by an HA tag (sPC-1; Figure 1A, Table 1; Salehi-Najafabadi et al., 2017; Zhu et al., 2011). The HA tag allowed us to quantify sPC-1 plasma membrane expression by measuring surface staining following addition of anti-HA antibodies to non-permeabilized cells. Co-expression of sPC-1 and PC-2 resulted in strong HA surface staining, whereas expression of sPC-1 alone resulted in only marginal surface staining, in both HEK293 cells (Figure 1B and C) and IMCD3 cells (Figure 1—figure supplement 1D and E). Co-expression of PC-2 and PC-1 with an extracellular HA tag but without the endogenous leader sequence (HAPC-1) failed to generate comparable HA surface staining, supporting the notion that an Ig κ-secretion sequence promotes membrane localization of the complex (Figure 1B). To test whether PC-2 facilitates sPC-1 membrane localization by promoting ER exit or by co-migration to the plasma membrane, we inserted a FLAG epitope into the extracellular tetragonal opening for polycystins (TOP) domain of PC-2 (PC-2FLAG) (Figure 1—figure supplement 1A, Table 1). Robust surface HA and FLAG staining only occurred when both sPC-1 and PC-2FLAG were co-expressed, suggesting that they are shuttled simultaneously to the plasma membrane (Figure 1—figure supplement 1B and C). Similarly, co-expression of sPC-1 with PC-2 containing a recently in an oocyte expression system characterized gain of function mutation of PC-2 (PC-2F604P, Table 1; Arif Pavel et al., 2016) resulted in comparable HA surface expression (Figure 1B and C). Figure 1 with 1 supplement see all Download asset Open asset PC-1 and PC-2 form functional channels in the plasma membrane. (A) Illustration of PC-1 and PC-2F604P topology highlighting extracellular HA and secretion sequence (indicated with an s prefix) in PC-1. (B) Expression and immunostaining of HEK293 cells transfected with sPC-1/PC-2, HAPC-1/PC-2 with endogenous leader sequence, sPC-1/PC-2F604P, and sPC-1 alone. Left column, representative images of HA surface staining (see methods). Middle column, total staining for PC-2 or PC-1. Right column, merged images. Scale bar: 10 μm. (C) Quantification of relative anti-HA surface staining shown in B. ***: p<0.0001. (D) Representative currents from each construct recorded in the whole cell patch clamp configuration in response to voltage step pulses (left). (E) Whole-cell I-V relationship of sPC-1/PC-2F604P (n = 6, red) and PC-1/PC-2F604P (n = 10, white). Arrow indicates the expanded view of I-V relationship between membrane potentials of −50 mV and +50 mV. (F) Current densities for sPC-1/PC-2F604P, HAPC-1/PC-2F604P, sPC-1/PC-2, and PC-2F604P alone at +100 mV. (G) Relative open probability of sPC-1/PC-2F604P at −80 mV calculated using a Boltzmann distribution fitted to I/Imin of tail current amplitudes, black line (n = 6, red). (H) Top view of the human PC-1/PC-2 polycystin complex using pymol (Su et al., 2018). Positively charged R4100, R4107, and H4111 are highlighted in red and displayed in stick shape (top). Side view of selectivity filter showing the location of three positively charged amino acids on PC-1 TM-11 (bottom). (I) Representative whole cell currents of sPC-1pore/PC-2F604P. (J) I-V relationship of sPC-1/PC-2F604P (n = 6, red) and sPC-1pore/PC-2F604P (n = 6, blue). Arrow indicates the expanded view of I-V relationship between −50 mV and +50 mV. Current densities of sPC-1/PC-2F604P and sPC-1pore/PC-2F604P were compared at +100 mV holding potential. (K) Relative open probability analysis of sPC-1/PC-2F604P (n = 6, red) and sPC-1pore/PC-2F604P (n = 6, blue) during a −80 mV tail pulse. Statistical significance is indicated by the background. ***p<0.001 (dark gray), **p<0.01 (gray), *p<0.05 (light gray). Statistical analysis was computed using student t-test unpaired one-pair. Table 1 Constructs used in this study. Construct nameProtein tagMutationsPC-1/PC-2F604Pκ-IgG signal peptide and HA tag at 5' end of PC-1F604P in PC-2PC-1/PC-2F604PHA tag at 5' end of PC-1F604P in PC-2sPC-1/PC-2κ-IgG signal peptide and HA tag at 5' end of PC-1wild type PC-2sPC-1/PC-2FLAGκ-IgG signal peptide and HA tag at 5' end of PC-1, FLAG insertion in PC-2 TOP domain (pos. 904):…PSNGT-DYKDDDK-SFIFY…PC-2F604P-F604P in PC-2sPC-1pore/PC-2F604Pκ-IgG signal peptide and HA tag at 5' end of PC-1R4100G, R4107G, H4111G in PC-1 F604P in PC-2PC-1ΔNT/PC-2HA tag (P2Y12)Substitution of PC-1 N-terminus adjacent to TM-1 (position 9184) with P2Y12 N-terminus. (HA_P2Y12_mypydvpdyaqavdnltsapgntslctrdykitq)-(vrfvfp…_PC-1)PC-1ΔNT/PC-2F604PHA tag (P2Y12)Substitution of PC-1 N-terminus with P2Y12 N-terminus F604P in PC-2sPC-1L3/PC-2κ-IgG signal peptide and HA tag at 5' end of mPC-1L3-sPC-1L31NT/PC-2κ-IgG signal peptide and HA tag at 5' end of PC-1 N-terminusSubstitution of mPC-1L3 N-terminus with hPC-1 N-terminus at GPS cleavage (hPC-1…CLTR-HLTFFSS…mPC-1L3)sPC-11L3NT/PC-2κ-IgG signal peptide and HA tag at 5' end of PC-1L3 N-terminusSubstitution of hPC-1 N-terminus with 1-1052aa of mPC-1L3 at GPS cleavage (mPC-1L3…QCLCD-HLTAFGA…hPC-1)sPC-11L3CTL/PC-2F604Pκ-IgG signal peptide and HA tag at 5' end of PC-1Replacement of PC-1 C-type lectin domain (407-547aa) with C-type lectin domain of PC-1L3 (30-150aa)sPC-1L31CTL/PC-2F604Pκ-IgG signal peptide and HA tag at 5' end of PC-1L3Replacement of PC-1L3 C-type lectin domain (30-138aa) with C-type lectin domain of PC-1 (415-535aa)sPC-1/PC-2F604PN375Qκ-IgG signal peptide and HA tag at 5' end of PC-1 N-terminusF604P in PC-2, N375Q in PC-2 Having established robust plasma membrane expression of sPC-1/PC-2 and sPC-1/PC-2F604P, we asked whether these proteins form functional channels using whole cell patch clamp recordings. Interestingly, only sPC-1/PC-2F604P produced constitutively active, outward rectifying currents (122.6 ± 23.0 pA/pF at +100 mV, n = 7) with a half-maximal activation voltage of +112.5 mV (Figure 1D, E and G). sPC-1/PC-2 (5.1 ± 1.0 pA/pF, n = 11), HAPC-1/PC-2F604P (5.0 ± 2.3 pA/pF, n = 15) and PC-2F604P alone (6.4 ± 2.1 pA/pF, n = 12) yielded negligible currents at +100 mV (Figure 1F). To investigate the functional contribution of PC-1 to heteromeric polycystin complexes, we mutated three positively charged amino acids in the putative ion permeation pathway to the small, uncharged amino acid glycine (R4100G, R4107G, and H4111G) in order to facilitate cation permeation (sPC-1pore; Figure 1H, Table 1). In combination with PC-2F604P, currents of 71.1 ± 5.4 pA/pF (n = 6) were generated at +180 mV (sPC-1pore/PC-2F604P; Figure 1I). Although the recorded current density was ~50 pA/pF smaller than for sPC-1/PC-2F604P (Figure 1J), the relative open probability at −100 mV was higher (0.3 ± 0.0 for sPC-1pore/PC-2F604P; 0.1 ± 0.0 for sPC-1/PC-2F604P), suggesting that the pore mutant resides in a more open state (Figure 1K). Collectively, these results demonstrate that PC-1/PC-2 subunits form a functional channel when reconstituted in mammalian cells and support the notion that PC-1 subunits form part of the core channel complex. The PC-1 N-terminus is essential for polycystin complex activation We sought to determine whether PC-1 subunits are involved in the regulation of the polycystin complex by investigating the functional role of the N-terminal domain. We reasoned that the N-terminus must be a critical determinant of polycystin function because ADPKD-causing mutations occur with high frequency in this region and therefore investigated a family member with a divergent N-terminus, PC-1L3 (Figure 2A and Figure 2—figure supplement 1A). Substitution of PC-1L3's signal peptide with the κ IgG secretion sequence (sPC-1L3, Table 1) resulted in strong surface HA staining when co-expressed with PC-2 or PC-2F604P (Figure 2B and D). However, we were unable to record convincing currents in sPC-1L3/PC-2F604P-transfected HEK cells (3.5 ± 0.0 pA/pF, n = 12), suggesting that, although present in the plasma membrane, the complex is inactive (Figure 2C). To determine the structural domains in PC-1 subunits that are required for channel activation, we generated chimeras between PC-1 and PC-1L3: the N-terminus of PC-1 fused to the 11-TM core of PC-1L3 preceding the GPS cleavage site (sPC-1L31NT) and the opposing arrangement (sPC-11L3NT) (Figure 2A, Table 1). We also substituted the entire PC-1 N-terminus with the 26 amino acid-long N-terminus of the P2Y12 receptor and an extracellular HA tag (PC-1ΔNT, Table 1); a modification known to increase surface expression of GPCRs without impairing functionality (Liberles and Buck, 2006; Liebscher et al., 2015). Figure 2 with 1 supplement see all Download asset Open asset The PC-1 N-terminus is essential for polycystin complex activation. (A) Schematic diagram illustrating topology of PC-1 (blue) and PC-1L3 (yellow), including PC-1ΔNT (top), PC-1 N-terminal chimeras (top), and PC-1L3 N-terminal chimeras (bottom). (B) Immunostaining of HEK293 cells transfected with indicated chimera. Left, representative images of anti-HA surface staining. Middle, total staining for PC-2. Right, merged images. Scale bar: 10 μm. (C) Representative whole cell current traces of each construct obtained from the voltage step pulses shown in Figure 1D. (D) Quantification of anti-HA surface staining of each construct shown in (B). ***p<0.001. (E) I-V relationships for N-terminal chimeras sPC-1L31NT/PC-2F604P (n = 5, purple), sPC-11L3CTL/PC-2F604P (n = 10, light blue) and sPC-1L3/PC-2F604P (n = 12, orange). (F) Relative open probabilities for sPC-1L31NT/PC-2F604P (n = 5, purple) and sPC-1/PC-2F604P (n = 6, red, dataset from Figure 1E) obtained during tail pulses at −80 mV. Statistical significance is indicated by the background. ***p<0.001 (dark gray), **p<0.01 (gray), *p<0.05 (light gray). Statistical analysis was computed using student t-test unpaired one-pair. (G) I-V relationships for sPC-1/PC-2F604P (n = 6, red), sPC-1L31CTL/PC-2F604P (n = 5, green), and sPC-1L3/PC-2F604P (n = 12, black). All chimeras localized to the plasma membrane when co-expressed with PC-2F604P (Figure 2B and D). PC-1ΔNT resulted in the strongest surface staining, likely due to the smaller size of this protein. Of the three chimeras, only sPC-1L31NT/PC-2F604P generated small but appreciable outward currents (35.9 ± 12.9 pA/pF, n = 5). Currents in cells expressing sPC-11L3NT/PC-2F604P (9.1 ± 1.9 pA/pF, n = 10) and PC-1ΔNT/PC-2F604P (7.6 ± 1.5 pA/pF, n = 10) were negligible (Figure 2C and E). Furthermore, the relative open probability of the sPC-1L31NT/PC-2F604P chimera at −100 mV (0.44 ± 0.0) was ~four-fold higher than sPC-1/PC-2F604P (0.1 ± 0.0) and similar to that for sPC-1pore/PC-2F604P (0.3 ± 0.0) (Figure 2F). Interestingly, the predicted ion permeation region of PC-1L3 does not contain the positively charged amino acids found in PC-1, offering a possible explanation for the greater open probability observed in the sPC-1L31NT/PC-2F604P chimeras (Figure 2—figure supplement 1A). In terms of channel regulation, these results support the hypothesis that the N-terminus of PC-1 is a key determinant of polycystin complex activation. Polycystin activation depends on CTL and TOP domains To gain a more detailed understanding of the role played by the N-terminus in polycystin complex activation, we replaced smaller regions of the PC-1L3 N-terminus with those from PC-1. One chimera replaced the 108 AA CTL domain of PC-1L3 with the CTL of PC-1 (sPC-1L31CTL; Figure 2A, Table 1). sPC-1L31CTL/PC-2F604P generated robust outward currents (87.5 ± 9.0 pA/pF, n = 5) (Figure 2C and G) whereas the inverse chimera (sPC-11L3CTL) produced only minimal currents (9.1 ± 1.9 pA/pF, n = 10). These data strongly suggest that the PC-1 CTL domain is essential to activate the polycystin complex. In mammalian cells, the PC-1 ectodomain undergoes at least one proteolytic cleavage event at the GPS site, suggesting that the N-terminus might be liberated as a secreted ligand. Indeed, it has been reported that PC-1 N-terminus-containing exosomes interact with primary cilia in kidney nephrons (Hogan et al., 2009). We therefore investigated whether fragments of the PC-1 N-terminus can regulate the polycystin complex. We purified various PC-1 NTFs from HEK cell supernatant (Figure 2—figure supplement 1C–1E) and tested their ability to confer channel activity on PC-1ΔNT/PC-2F604P heteromers when included in the bath solution. Surprisingly, application of an NTF spanning the distal region AA 24–852 (NTF24-852), including the CTL domain between PKD I and II (amino acids 415–531; Figure 2—figure supplement 1B and C), conferred the ability to carry outward currents on PC-1ΔNT/PC-2F604P heteromers (65.5 ± 37.2 pA/pF, n = 6) (Figure 3A and C). Similarly, a shorter NTF containing the CTL domain (NTF263-535; Figure 2—figure supplement 1B and C) was also able to induce outward currents (61.4 ± 10.5 pA/pF, n = 4; Figures 3A, B and C). In both cases, the currents were smaller than those for sPC-1/PC-2F604P (Figure 1F). Boiling NTF263-535 at 95°C for 10 min rendered it inactive (Figure 3B). Next, we perfused HEK cells overexpressing PC-1ΔNT/PC-2F604P heteromers with NTF263-535 and measured acute changes in current density. While PC-1ΔNT/PC-2F604P channels remained inactive in the absence of NTF263-535, acute application increased current density by 765.1 ± 185.05% (from 5.9 ± 1.3 pA/pF to 59.1 ± 19.3 pA/pF, n = 7) within 10 s of application (Figures 3D, F and G). However, non-transfected cells did not respond to NTF263-535 under comparable condition (4.6 ± 1.6 pA/pF to 6.8 ± 1.4 pA/pF, n = 9) (Figure 3E and G). We then asked whether the removal of PC-1 N-terminus inactivates the polycystin heteromer. To test this possibility, we perfused sPC-1/PC-2F604P overexpressing HEK cells with 0.125% trypsin, assuming that proteolytic digestion of extracellular proteins also removes critical regions within the PC-1 N-terminus. As shown in Figure 3H perfusion with 0.125% trypsin decreased sPC-1/PC-2F604P dependent currents by 78.4 ± 4.9% (from 52.0 ± 17.1 pA/pF to 8.9 ± 1.4 pA/pF, n = 6, Figure 3H and J, and K). By contrast, untransfected cells did not respond to 0.125% trypsin (7.2 ± 1.9 pA/pF versus 4.6 ± 0.8 pA/pF, n = 5). Taken together, these data suggest that the N-terminus including the CTL of PC-1 is necessary and sufficient to activate the polycystin complex. Figure 3 Download asset Open asset Polycystin activation depends on the C-Type lectin domain. (A) Representative whole cell currents of PC-1ΔNT/PC-2F604P after application of PC-1 NTF24-852 (left) or NTF263-535 (right). (B) I-V relationships of PC-1ΔNT/PC-2F604P with addition of NTF263-535 (n = 6, pink), PC-1ΔNT/PC-2F604P with addition of heat-inactivated fragment (n = 8, black), and PC1ΔNT/PC-2F604P alone (n = 8, green). (C) Current densities of untransfected cells (gray) and PC-1ΔNT/PC-2F604P after addition of control medium (n = 7, white), NTF24-852 (n = 5, blue) and NTF263-535, (n = 6, pink). ***, p<0.001. (D) Time course of PC-1ΔNT/PC-2F604P transfected cells in response to NTF263-535. Currents are recorded at +180 mV (black) and −100 mV (red). Orange bar indicates addition of NTF. (E) Time course of untransfected cells in response to NTF263-535. Conditions are identical to D. (F) I-V relationships obtained by voltage ramp pulse for PC-1ΔNT/PC-2F604P before (black) or after (red) NTF263-535 application. (G) Current densities at +100 mV potential from PC-1ΔNT/PC-2F604P (n = 7) or untransfected cells (n = 9) before (blank) or after application (red) of NTF263-535. (H) Time course of sPC-1/PC-2F604P transfected cells in response to application of 0.125% trypsin. Currents are recorded at +180 mV (black) and −100 mV (red). Orange bar indicates addition of trypsin. (J) I-V relationships for sPC-1/PC-2F604P before (black) and after (red) 0.125% trypsin application. (K) Current densities at +100 mV potential from for sPC-1/PC-2F604P (n = 6) and untransfected cells (n = 5) before (blank) or after (red) 0.125% trypsin application. Next, we asked how the extracellular CTL domain in PC-1 might regulate activation of the entire channel complex. We reasoned that the TOP domain in PC-2 is well positioned to function as a surface receptor as it forms a bridge between the pore domain and voltage sensor-like domain, and is critical for channel activation (Shen et al., 2016; Vien et al., 2020). In addition, different glycosylation patterns of the TOP domain might underlie conformational changes in the homomeric PC-2 complex (Wilkes et al., 2017). We observed prominent glycosylation of the TOP domain in PC-2 at position N375, N362, and N328 (Figure 4A). Moreover, although mutation of N375 to glutamine did not affect surface localization of sPC-1/PC-2F604PN375Q (Figure 4B and C), it rendered the channel inactive (Figure 4D and E). Thus, glycosylation of the TOP domain in PC-2 appears to be required for CTL-dependent polycystin activation. Figure 4 Download asset Open asset C-type lectin binds to the TOP domain of PC-2. (A) Location of carbohydrates (yellow mesh) bound to N375 in the TOP domain of the PC-2 channel (protein data bank ID: 5T4D). Right, expanded view of carbohydrate binding to N375 site. (B) Quantification of anti-HA surface staining of sPC-1/PC-2F604P and sPC-1/PC-2F604PN375Q compared to untransfected cells. ***p<0.001. (C) Immunostaining of HEK293 cells transfected with sPC-1/PC-2F604PN375Q. Scale bar: 10 μm. (D) Representative whole cell current traces of sPC-1/PC-2F604PN375Q. (E) I-V relationships for sPC-1/PC-2F604PN375Q (n = 11, orange) and sPC-1/PC-2F604P (n = 6, red, dataset from Figure 1E). N-terminal PC-1 fragments activate ciliary polycystin complexes Although the results so far strongly suggest that the PC-1 CTL domain is critical for activating heterologous polycystin complexes, native polycystin complexes reside in the specialized membranes of primary cilia (Liu et al., 2018; Kleene and Kleene, 2017; Vien et al., 2020). We, therefore, investigated whether the PC-1 N-terminus is able to activate polycystin complexes in the cilia of polarized mouse epithelial (mIMCD-3) cells using excised ciliary membrane patch clamp recordings (Figure 5—figure supplement 1B). Without any NTFs, we readily detected single channel openings at potentials more positive than +60 mV and more negative than −80 mV (Figure 5A) in 7 out of 38 cilia. After ablating PC-1 expression in mIMCD3 cells using CRISPR/CAS9 (Figure 5—figure supplement 1A) ciliary channels remained readily detectable in 4 of 13 cilia (Figure 5B), in agreement with previous reports (Liu et al., 2018). In addition, single channel conductance was comparable between wt mIMCD3 (γ = 79.5 ± 0.1 pS) and PC-1 knockout cilia (γ = 82.3 ± 0.0 pS) (Figure 5—figure supplements 1G, H, 2A and B). However, we noted that the open probability of channels at negative membrane potentials in PC-1 knock out primary cilia (0.58 ± 0.01) was higher than in wt IMCD3 cilia (0.08 ± 0.01) (Figure 5G). These data suggest that PC-2 forms a homomeric complex in the absence of PC-1 subunits with a higher open probability than that of PC-1/PC-2 heteromers. The results also support the idea that PC-1 subunits impede cation flux across the membrane, in agreement with our PC-1 pore mutant data (Figure 1I–K). Ablation of PC-2 expression using CRISPR/CAS9 rendered primary cilia electrically silent (n = 11), confirming that PC-2 subunits are an essential component of polycystin channels (Figure 5C; Liu et al., 2018; Kleene and Kleene, 2017). Figure 5 with 2 supplements see all Download asset Open asset An N-terminal PC-1 fragment activates ciliary polycystin complexes. (A, B, C) Excised ciliary inside-out patch clamp recordings of channels from mIMCD-3 cells (left), PC-1 knockout mIMCD-3 cells (middle), and PC-2 knockout mIMCD-3 cells (right). (D) Single channel patch clamp recordings at −100 mV in cell attached configuration for sPC-1/PC-2F604P (top) and excised inside-out ciliary patches for mIMCD-3, mIMCD-3 with addition of NTF263-535 in pipette solution, and PC-1 knockout cells (rows 2–4, respectively). (E) Open time histogram of channel openings at −100 mV holding potential. (F) Time constants of open time distributions in E obtained using one decay equation, *p<0.05 (n = 7/38 for mIMCD-3 cilia, n = 4/13 for PC-1 KO, n = 4/23 for mIMCD-3 cilia + NTF263-535, and n = 6 for sPC-1/PC-2F604P). (G) Absolute open probabilities for recordings shown in A, B, and C, **p<0.01. To test whether NTFs can activate the native polycystin complex, we recorded single channels in inside out patches pulled from primary cilia in the presence of 0.7 μg/mL (equal to ~50 nM final concentration) of NTF263-535 in the recording pipette. Although the percentage of electrically active cilia (n = 4/13) remained largely unaffected, NTF263-535 increased the probability of single channels openings by ~five-fold and significantly prolonged the duration of channel openings at negative membrane potentials (Figure 5E–G). Collectively, these findings demonstrate that PC-2 subunits are essential for functional

Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.