The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant ~600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.

Neuronal dendrites must relay synaptic inputs over long distances, but the mechanisms by which activity-evoked intracellular signals propagate over macroscopic distances remain unclear. Here, we discovered a system of periodically arranged endoplasmic reticulum-plasma membrane (ER-PM) junctions tiling the plasma membrane of dendrites at ∼1 μm intervals, interlinked by a meshwork of ER tubules patterned in a ladder-like array. Populated with Junctophilin-linked plasma membrane voltage-gated Ca2+ channels and ER Ca2+-release channels (ryanodine receptors), ER-PM junctions are hubs for ER-PM crosstalk, fine-tuning of Ca2+ homeostasis, and local activation of the Ca2+/calmodulin-dependent protein kinase II. Local spine stimulation activates the Ca2+ modulatory machinery, facilitating signal transmission and ryanodine-receptor-dependent Ca2+ release at ER-PM junctions over 20 μm away. Thus, interconnected ER-PM junctions support signal propagation and Ca2+ release from the spine-adjacent ER. The capacity of this subcellular architecture to modify both local and distant membrane-proximal biochemistry potentially contributes to dendritic computations.

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].

Excipients are often the major component of the formulation that critically affect the dosage form, manufacturing process, product performance, stability and safety. They exert different roles and functions in a dosage form. Selecting excipients with appropriate safety and tolerability is a major hurdle in paediatric formulation development. The suitability of a particular excipient will be dependent on the context of its use with regard to the paediatric age range, acute versus chronic use, and clinical risk-benefit of the disease, active and excipient. Scientists are encouraged to apply the principle of risk-benefit to assess the suitability of excipients to the specific paediatric population. Indicative list of parameters that should be taken into consideration and hierarchy of information sources when assessing the excipients risks is provided by regulatory agencies. However, the approach to be taken and details of how the risk evaluation should be undertaken are lacking. There is a need for a systematic approach to selection of excipients and assessment of the risk of excipient exposure. The Paediatric Excipients Risk Assessment (PERA) framework developed and proposed in this paper provides a structured, systematic decision-making framework via customizable tools and processes that can help to improve the transparency and communications on the selection and justification of use of excipients in a paediatric formulation.

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φu < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φu > 20%─a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

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 severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a’s ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

AIM:The EMA defines acceptability as "the overall ability and willingness of the patient to use, and their caregiver to administer, the medicine as intended" [1]. This paper seeks to outline issues of acceptability in relation to injectable therapy, namely intravenous (IV), intramuscular (IM) and subcutaneous (SC) administration routes, and to lay a foundation to identify a minimum set of data that would satisfy Regulatory Authorities when discussing the acceptability of an injectable product. In addition, it will alert drug product developers to other factors that might contribute to good practice, alternative administration strategies and overall adherence to achieve successful treatment. Whilst the term 'parenteral' means "outside the intestine" [2,3] and so potentially covers a range of administration routes including intranasal and percutaneous administration, this review focuses on IV, IM and SC administration by injection. The use of indwelling canulae or catheters to reduce venepuncture and facilitate prolonged treatment is common and may impact acceptability [4]. This may be influenced by information provided by the manufacturer but is not always in their direct control. Other injectable products suitable for routes such as intradermal, intra-articular, intraosseous and intrathecal, share the requirement to be acceptable but are not specifically covered in this paper [2,5].

The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

CatSper channels (CatSper1-4, nomenclature as agreed by NC-IUPHAR [14]) are putative 6TM, voltage-gated, alkalinization-activated calcium permeant channels that are presumed to assemble as a tetramer of α-like subunits and mediate the current ICatSper [23]. In mammals, CatSper subunits are structurally most closely related to individual domains of voltage-activated calcium channels (Cav) [40]. CatSper1 [40], CatSper2 [37] and CatSpers 3 and 4 [27, 21, 36], in common with a putative 2TM auxiliary CatSperβ protein [26] and two putative 1TM associated CatSperγ and CatSperδ proteins [46, 12], are restricted to the testis and localised to the principle piece of sperm tail. The novel cross-species CatSper channel inhibitor, RU1968, has been proposed as a useful tool to aid characterisation of native CatSper channels [41].Two-pore channels (TPCs) are structurally related to CatSpers, CaVs and NaVs. TPCs have a 2x6TM structure with twice the number of TMs of CatSpers and half that of CaVs. There are three animal TPCs (TPC1-TPC3). Humans have TPC1 and TPC2, but not TPC3. TPC1 and TPC2 are localized in endosomes and lysosomes [5]. TPC3 is also found on the plasma membrane and forms a voltage-activated, non-inactivating Na+ channel [6]. All the three TPCs are Na+-selective under whole-cell or whole-organelle patch clamp recording [48, 8, 7]. The channels may also conduct Ca2+ [31].

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]).

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state.

Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals.

The 2TM domain family of K channels are also known as the inward-rectifier K channel family. This family includes the strong inward-rectifier K channels (Kir2.x) that are constitutively active, the G-protein-activated inward-rectifier K channels (Kir3.x) and the ATP-sensitive K channels (Kir6.x, which combine with sulphonylurea receptors (SUR1-3)). The pore-forming α subunits form tetramers, and heteromeric channels may be formed within subfamilies (e.g. Kir3.2 with Kir3.3).

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.