TRPM5 is a Ca2+-activated monovalent cation channel essential for taste perception, insulin secretion and gastrointestinal chemosensation. Canonical TRPM5 activation requires Ca2+ binding at two distinct sites: an agonist site within the lower vestibule of the S1–S4 pocket in the transmembrane domain (CaTMD) and a modulatory site in the intracellular domain (CaICD) that tunes voltage dependence and agonist sensitivity. Here we characterize CBTA as a noncalcium agonist that binds to the upper vestibule of the S1–S4 pocket, directly above CaTMD. CBTA alone mimics the dual role of CaTMD and CaICD, merging agonist activation with voltage modulation. CBTA also renders TRPM5 supersensitive to Ca2+, synergistically hyperactivating the channel even at near-resting Ca2+ levels. We further demonstrate that the inhibitor triphenylphosphine oxide binds the same site but stabilizes a nonconductive state. These opposing effects reveal the upper S1–S4 pocket as a multifunctional regulatory hub integrating activation, inhibition and modulation in TRPM5. TRPM5 activation requires Ca2+ binding at an S1–S4 pocket in the transmembrane domain (TMD) and a modulatory site in the intracellular domain (ICD). Structural and functional analyses of TRPM5 with Ca2+, the agonist CBTA and the inhibitor triphenylphosphine oxide—all engaging the S1–S4 pocket—reveal how modulation of channel activity is communicated across the TMD and ICD.

Detecting noxious heat is vital for survival, triggering protective pain responses. The TRPM3 channel is a key nociceptor and a promising therapeutic target for pain and neurological disorders. Here we show that the rabbit TRPM3 is intrinsically dynamic, with its intracellular domain (ICD) sampling both resting and activated states, but favoring the resting state in the absence of stimulation. We reveal that heat and the synthetic agonist CIM0216 shift the equilibrium toward activation by inducing a similar ICD rearrangement. Mutations that facilitate ICD movement enhance sensitivity to both thermal and chemical stimuli, underscoring the central role of the ICD in channel gating. We also show that the antagonist primidone binds the same site as CIM0216 in the S1-S4 domain but inhibits channel activation. This study provides a structural framework for a mechanistic understanding of thermal and chemical gating of TRPM3 and for guiding the rational design of TRPM3-targeted analgesics and neurotherapeutics.

Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above, which were used to identify endogenous PANX1 phosphorylation at these two sites, are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.

Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species. Using time-resolved cryo-EM, the authors capture complete structural snapshots of the enzymatic cycle coupled with channel gating in a TRPM-type channel enzyme.

Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by PACC1 (TMEM206). PAC regulates endosomal acidification and macropinosome shrinkage by releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP2) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP2 at pH 4.0 and identified its putative binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP2-binding. Structural and electrophysiological analyses suggest that PIP2 inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP2 as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.

TRPM2 is a calcium-permeable, non-selective cation channel present in different species from unicellular Choanoflagellates to human. TRPM2 plays an important role in the survival of early species and is critically involved in diverse physiological processes including core body temperature regulation, immune response, insulin secretion, and apoptosis. TRPM2 is polymodal and can be activated by a wide range of stimuli including warm temperature, oxidative stress and NAD+-related metabolites such as ADP-ribose (ADPR). The consensus was that in the presence of calcium, ADPR activates TRPM2 upon binding to its characteristic C-terminal NUDT9-H domain. However, recent studies by our group and others have established that the N-terminal MHR1/2 domain contains a previously unknown ADPR binding site that is conserved across species and is absolutely essential for TRPM2 gating (Kühn et al., 2016, 2019; Huang et al., 2018, 2019; Tóth et al., 2020). The important role of MHR1/2 domain in channel gating is further supported by the fact that the antagonist 8-Br-cADPR inhibits the channel by binding to the MHR1/2 domain and stabilizing the channel in an apo-like conformation (Kolisek et al., 2005; Huang et al., 2019). In contrast to the conserved role of the MHR1/2 domain, the function of NUDT9-H domain has changed with evolution. In invertebrates, the NUDT9-H domain does not directly gate the channel but is indirectly involved in channel gating by hydrolyzing the agonist ADPR into AMP and ribose-5-phosphate (Kühn et al., 2016; Iordanov et al., 2019). In vertebrates, the NUDT9-H domain has no enzymatic activity, but cooperates with the MHR1/2 domain to open the channel (Iordanov et al., 2016; Huang et al., 2018, 2019).

PAC channel is an evolutionarily conserved pH-gated chloride ion channel. Its activity at the plasma membrane is widely observed across different tissues. Under pathological conditions associated with acidosis, such as ischemic stroke, PAC at the cell surface is activated by a drop in tissue pH, allowing the entry of chloride into the cells, which in turn causes cell swelling and contributes to acid-induced brain injury. PAC also traffics to endosomes, where it regulates lumen acidification. Threshold for PAC activation depends on its subcellular localization, so we hypothesized that PAC channel is regulated by phosphatidylinositol PI(4,5)P2 lipid that is exclusively present in the plasma membrane.

The proton-activated chloride (PAC) channel plays a key role in determining the acid toxicity to mammalian cells. However, the mechanism underlies its pH-sensing function remains unclear. We analyzed the PAC channel in nanodisc using single-particle cryo-electron microscopy (cryo-EM) and revealed structures of PAC at two different pH conditions (pH8 and pH4). The homo-trimeric PAC channel contains a transmembrane domain (TMD) with two helices and an extracellular domain (ECD). Environmental acidification induces a major conformational rearrangement in the TMD and TMD-ECD interface, including a compression dynamics of the channel complex and a domain-swapping movement of the TM1.

The Ca 2+ -activated TRPM5 channel plays an essential role in the perception of sweet, bitter, and umami stimuli in type II taste cells and in insulin secretion by pancreatic beta cells 1–3 . Interestingly, the voltage dependence of TRPM5 in taste bud cells depends on the intracellular Ca 2+ concentration 4 , yet the mechanism remains elusive. Here we report cryo-electron microscopy structures of the zebrafish TRPM5 in an apo closed state, a Ca 2+ -bound open state, and an antagonist-bound inhibited state, at resolutions up to 2.3 Å. We defined two novel ligand binding sites: a Ca 2+ binding site (Ca ICD ) in the intracellular domain (ICD), and an antagonist binding site in the transmembrane domain (TMD) for a drug (NDNA) that regulates insulin and GLP-1 release 5 . The Ca ICD site is unique to TRPM5 and has two roles: shifting the voltage dependence toward negative membrane potential, and promoting Ca 2+ binding to the Ca TMD site that is conserved throughout Ca 2+ -sensitive TRPM channels 6 . Replacing glutamate 337 in the Ca ICD site with an alanine not only abolished Ca 2+ binding to Ca ICD but also reduced Ca 2+ binding affinity to Ca TMD , suggesting a cooperativity between the two sites. We have defined mechanisms underlying channel activation and inhibition. Conformational changes initialized from both Ca 2+ sites, 70 Å apart, are propagated to the ICD–TMD interface and cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents to treat metabolic disorders.

Pannexin 1 (PANX1) is a large-pore ion channel that plays important roles in various biological processes such as apoptotic cell clearance, inflammation, and blood pressure regulation. However, a detailed mechanistic understanding of PANX1 channel function remains elusive due to the lack of atomic structures. In this study, we performed single-particle cryo-electron microscopy (cryo-EM) analysis combined with electrophysiology and computational simulations to elucidate the channel gating, ion permeation, and drug inhibition mechanisms. Our study revealed a heptameric PANX1 channel assembly that conducts ions through two distinct pathways. In normal cellular conditions, chloride ions permeate mainly through the narrow side tunnels formed by the intracellular domain of adjacent PANX1 subunits. During apoptosis, caspase protease irreversibly cleaves the C-terminal tail of PANX1, opening the central ion-conducting pore. We identified the carbenoxolone (CBX) as a channel blocker by interacting with the extracellular exit of PANX1. We also obtained a gap-junction like structure from a glycosylation-deficient mutant (N255A), supporting the notion that extracellular glycosylation is the key determinant of PANX1 gap-junction formation. In sum, our structural and functional analyses provide a foundation to investigate the regulatory mechanisms of PANX1, which can be further exploited for pharmaceutical applications.

The TRPM (transient receptor potential melastatin) family belongs to the superfamily of TRP cation channels. The TRPM subfamily is composed of eight members that are involved in diverse biological functions such as temperature sensing, inflammation, insulin secretion, and redox sensing. Since the first cloning of TRPM1 in 1998, tremendous progress has been made uncovering the function, structure, and pharmacology of this family. Complete structures of TRPM2, TRPM4, and TRPM8, as well as a partial structure of TRPM7, have been determined by cryo-EM, providing insights into their channel assembly, ion permeation, gating mechanisms, and structural pharmacology. Here we summarize the current knowledge about channel structure, emphasizing general features and principles of the structure of TRPM channels discovered since 2017. We also discuss some of the key unresolved issues in the field, including the molecular mechanisms underlying voltage and temperature dependence, as well as the functions of the TRPM channels' C-terminal domains.