Channel Family Research Articles

TMEM16F regulates pathologic α-synuclein secretion and spread in cellular and mouse models of Parkinson's disease.

One of the main hallmarks of Parkinson's disease (PD) pathology is the spread of the aggregate-prone protein α-synuclein (α-syn), which can be detected in the plasma and cerebrospinal fluid of patients as well as in the extracellular environment of neuronal cells. The secreted α-syn can exhibit "prion-like" behavior and transmission to naïve cells can promote conformational changes and pathology. The precise role of plasma membrane proteins in the pathologic process of α-syn is yet to be fully resolved. The TMEM16 family of lipid scramblases and ion channels has been recently associated with cancer and infectious diseases but is less known for its role in aging-related diseases. To elucidate the role of TMEM16F in α-syn spread, we transduced neurons derived from TMEM16F knockout mice with a reporter system that enables the distinction between donor and recipient neurons of pathologic α-synA53T. We found that the spread of α-synA53T was reduced in neurons derived from TMEM16F-knockout mice. These findings were recapitulated invivo in a mouse model of PD, where attenuated α-synA53T spread was observed when TMEM16F was ablated. Moreover, we identified a single nucleotide polymorphism in TMEM16F of Ashkenazi Jewish PD patients resulting in a missense Ala703Ser mutation with enhanced lipid scramblase activity. This mutation is associated with altered regulation of α-synA53T extracellular secretion in cellular models of PD. Our study highlights TMEM16F as a novel regulator of α-syn spread and as a potential therapeutic target in synucleinopathies.

Conservation of the cooling agent binding pocket within the TRPM subfamily.

Transient receptor potential (TRP) channels are a large and diverse family of tetrameric cation-selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca2+. Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here, we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4, and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of mouse TRPM4 to intracellular Ca2+, enhancing the sensitivity of the channel to Ca2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related ligand binding pockets that are allosterically coupled to opening of the pore.

Functional expression of the chimera proteins of Nav1.7 and NavAb in Escherichiacoli

Nav1.7 is a eukaryotic voltage-dependent Na channel (Nav) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Nav subtype are targeted in the development of Nav1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Nav1.7 inhibitors, the chimera protein (NavAb/1.7VSD) of Nav from prokaryotic Arcobacter butzleri (NavAb), in which extracellular loops of VSD were replaced with homologous loops from Nav1.7, serves as an effective model. This is because NavAb harbors only one VSD and the biological activity of NavAb/1.7VSD was previously confirmed. To date, NavAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of NavAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Nav1.7-specific peptide inhibitor. The results indicate that NavAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Nav1.7 inhibitors using the PERISS screening method.

Piezo1: the key regulators in central nervous system diseases.

The occurrence and development of central nervous system (CNS) diseases is a multi-factor and multi-gene pathological process, and their diagnosis and treatment have always posed a serious challenge in the medical field. Therefore, exploring the relevant factors in the pathogenesis of CNS and improving the diagnosis and treatment rates has become an urgent problem. Piezo1 is a recently discovered mechanosensitive ion channel that opens in response to mechanical stimuli. A number of previous studies have shown that the Piezo channel family plays a crucial role in CNS physiology and pathology, especially in diseases related to CNS development and mechanical stimulation. This article comprehensively describes the biological properties of Piezo1, focuses on the potential association between Piezo1 and CNS disorders, and explores the pharmacological roles of Piezo1 agonists and inhibitors in treating CNS disorders.

Research progress of two-pore potassium channel in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a secondary injury caused by restoring blood flow after acute myocardial infarction, which may lead to serious arrhythmia and heart damage. In recent years, the role of potassium channels in MIRI has attracted much attention, especially the members of the two-pore domain potassium (K2P) channel family. K2P channel has unique structure and function, and the formation of its heterodimer increases its functional diversity. This paper reviews the structural characteristics, types, expression and physiological functions of K2P channel in the heart. In particular, we pay attention to whether members of the subfamily such as TWIK, TREK, TASK, TALK, THIK and TRESK participate in MIRI and their related mechanisms. Future research will help to reveal the molecular mechanism of K2P channel in MIRI and provide new strategies for the treatment of cardiovascular diseases.

Biology, function and structure of the calcium homeostasis modulator family.

Calcium homeostasis modulators (CALHMs) are the most recently discovered members of the large-pore channel family. They mediate the conductance of ions and larger molecules, such as ATP, and play critical roles in pathways related to Alzheimer's disease, neuroinflammation, neuromodulation, taste perception and innate immune responses. Since the inaugural report on CALHM1 in 2008, significant breakthroughs have revealed their biological roles, ion and ATP channel functions, and structures, positioning the field for further advancements. In this review, we discuss the overall progress and recent developments in understanding the biological roles, functions and molecular structures of CALHM proteins.

Structural Basis for Voltage Gating and Dalfampridine Binding in the Shaker Potassium Channel.

The generation and propagation of action potentials in neurons depend on the coordinated activation of voltage-dependent sodium and potassium channels. Potassium channels of the Shaker family regulate neuronal excitability through voltage-dependent opening and closing of their ion conduction pore. This family of channels is an important therapeutic target, particularly in multiple sclerosis where the inhibitor dalfampridine (4-aminopyridine) is used to improve nerve conduction. The molecular details of how the voltage sensor domain drives opening of the pore domain has been limited by the lack of closed-state structures, also impairing the search for novel drugs. Using AlphaFold2-based conformational sampling methods we identify a structural model for the closed Shaker potassium channel where movement of the voltage sensor drives the opening trough interactions between the S4-S5 linker and S6 helix. We show experimentally that breakage of a backbone hydrogen bond is a critical part of the activation pathway. Through docking we identify a hydrophobic cavity formed by the pore domain helices that binds dalfampridine in the closed state. Our results demonstrate how voltage sensor movement drives pore opening and provide a structural framework for developing new therapeutic agents targeting the closed state. We anticipate this work will enable structure-based drug design efforts focused on state-dependent modulation of voltage-gated ion channels for the treatment of neurological disorders.

ω-Grammotoxin-SIA inhibits voltage-gated Na+ channel currents.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.

Implications of TRPM3 and TRPM8 for sensory neuron sensitisation.

Sensory neurons serve to receive and transmit a wide range of information about the conditions of the world around us as well as the external and internal state of our body. Sensitisation of these nerve cells, i.e.becoming more sensitive to stimuli or the emergence or intensification of spontaneous activity, for example in the context of inflammation or nerve injury, can lead to chronic diseases such as neuropathic pain. For many of these disorders there are only very limited treatment options and in order to find and establish new therapeutic approaches, research into the exact causes of sensitisation with the elucidation ofthe underlying mechanisms and the identification of themolecular components is therefore essential. These components include plasma membrane receptors and ion channels that are involved in signal reception and transmission. Members of the transient receptor potential (TRP) channel family are also expressed in sensory neurons and some of them play a crucial role in temperature perception. This review article focuses on the heat-sensitive TRPM3 andthe cold-sensitive TRPM8 (and TRPA1) channels and their importance in sensitisation of dorsal root ganglion sensory neurons is discussed based on studies related to inflammation and injury- as well as chemotherapy-induced neuropathy.

Functions of TRPs in retinal tissue in physiological and pathological conditions.

The Transient Receptor Potential (TRP) constitutes a family of channels subdivided into seven subfamilies: Ankyrin (TRPA), Canonical (TRPC), Melastatin (TRPM), Mucolipin (TRPML), no-mechano-potential C (TRPN), Polycystic (TRPP), and Vanilloid (TRPV). Although they are structurally similar to one another, the peculiarities of each subfamily are key to the response to stimuli and the signaling pathway that each one triggers. TRPs are non-selective cation channels, most of which are permeable to Ca2+, which is a well-established second messenger that modulates several intracellular signaling pathways and is involved in physiological and pathological conditions in various cell types. TRPs depolarize excitable cells by increasing the influx of Ca2+, Na+, and other cations. Most TRP families are activated by temperature variations, membrane stretching, or chemical agents and, therefore, are defined as polymodal channels. All TPRs are expressed, at some level, in the central nervous system (CNS) and ocular-related structures, such as the retina and optic nerve (ON), except the TRPP in the ON. TRPC, TRPM, TRPV, and TRPML are found in the retinal pigmented cells, whereas only TRPA1 and TRPM are detected in the uvea. Accordingly, several studies have focused on the search to unravel the role of TRPs in physiological and pathological conditions related to the eyes. Thus, this review aims to shed light on endogenous and exogenous modulators, triggered cell signaling pathways, and localization and roles of each subfamily of TRP channels in physiological and pathological conditions in the retina, optic nerve, and retinal pigmented epithelium of vertebrates.

The role of TRPV1 in chronic prostatitis: a review.

Chronic prostatitis is a prevalent male urinary system disorder characterized by pelvic discomfort or pain, bladder dysfunction, sexual dysfunction, and infertility. Pain and lower urinary tract symptoms (LUTS) are the most common symptoms, significantly impacting patients' quality of life and driving them to seek medical attention. Transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective calcium ion-dependent cation channel in the TRPV channel family that is widely distributed in neural tissue and plays a role in signal transmission. In this review, we provide a comprehensive overview of the current understanding of the role of TRPV1 in chronic prostatitis. The discussion focuses on the connection between TRPV1 and prostatitis pain and LUTS, and highlights the potential for targeting this channel in the development of novel treatment strategies.

PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK

The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.

Atomistic mechanisms of the regulation of small-conductance Ca2+-activated K+ channel (SK2) by PIP2

Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

TMC7 deficiency causes acrosome biogenesis defects and male infertility in mice.

Transmembrane channel-like (TMC) proteins are a highly conserved ion channel family consisting of eight members (TMC1-TMC8) in mammals. TMC1/2 are components of the mechanotransduction channel in hair cells, and mutations of TMC1/2 cause deafness in humans and mice. However, the physiological roles of other TMC proteins remain largely unknown. Here, we show that Tmc7 is specifically expressed in the testis and that it is required for acrosome biogenesis during spermatogenesis. Tmc7-/- mice exhibited abnormal sperm head, disorganized mitochondrial sheaths, and reduced number of elongating spermatids, similar to human oligo-astheno-teratozoospermia. We further demonstrate that TMC7 is colocalized with GM130 at the cis-Golgi region in round spermatids. TMC7 deficiency leads to aberrant Golgi morphology and impaired fusion of Golgi-derived vesicles to the developing acrosome. Moreover, upon loss of TMC7 intracellular ion homeostasis is impaired and ROS levels are increased, which in turn causes Golgi and endoplasmic reticulum stress. Taken together, these results suggest that TMC7 is required to maintain pH and ion homeostasis, which is needed for acrosome biogenesis. Our findings unveil a novel role for TMC7 in acrosome biogenesis during spermiogenesis.

Revealing a hidden conducting state by manipulating the intracellular domains in KV10.1 exposes the coupling between two gating mechanisms.

The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K+ channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1(KV10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.

Optimizing Nav1.7-Targeted Analgesics: Revealing Off-Target Effects of Spider Venom-Derived Peptide Toxins and Engineering Strategies for Improvement.

The inhibition of Nav1.7 is a promising strategy for the development of analgesic treatments. Spider venom-derived peptide toxins are recognized as significant sources of Nav1.7 inhibitors. However, their development has been impeded by limited selectivity. In this study, eight peptide toxins from three distinct spider venom Nav channel families demonstrated robust inhibition of hNav1.7, rKv4.2, and rKv4.3 (rKv4.2/4.3) currents, exhibiting a similar mode of action. The analysis of structure and function relationship revealed a significant overlap in the pharmacophore responsible for inhibiting hNav1.7 and rKv4.2 by HNTX-III, although Lys25 seems to play a more pivotal role in the inhibition of rKv4.2/4.3. Pharmacophore-guided rational design is employed for the development of an mGpTx1 analogue, mGpTx1-SA, which retains its inhibition of hNav1.7 while significantly reducing its inhibition of rKv4.2/4.3 and eliminating cardiotoxicity. Moreover, mGpTx1-SA demonstrates potent analgesic effects in both inflammatory and neuropathic pain models, accompanied by an improved in vivo safety profile. The results suggest that off-target inhibition of rKv4.2/4.3 by specific spider peptide toxins targeting hNav1.7 may arise from a conserved binding motif. This insight promises to facilitate the design of hNav1.7-specific analgesics, aimed at minimizing rKv4.2/4.3 inhibition and associated toxicity, thereby enhancing their suitability for therapeutic applications.

Identification of Shaker Potassium Channel Family Members and Functional Characterization of SsKAT1.1 in Stenotaphrum secundatum Suggest That SsKAT1.1 Contributes to Cold Resistance.

Stenotaphrum secundatum is an excellent shade-tolerant warm-season turfgrass. Its poor cold resistance severely limits its promotion and application in temperate regions. Mining cold resistance genes is highly important for the cultivation of cold-resistant Stenotaphrum secundatum. Although there have been many reports on the role of the Shaker potassium channel family under abiotic stress, such as drought and salt stress, there is still a lack of research on their role in cold resistance. In this study, the transcriptome database of Stenotaphrum secundatum was aligned with the whole genome of Setaria italica, and eight members of the Shaker potassium channel family in Stenotaphrum secundatum were identified and named SsKAT1.1, SsKAT1.2, SsKAT2.1, SsKAT2.2, SsAKT1.1, SsAKT2.1, SsAKT2.2, and SsKOR1. The KAT3-like gene, KOR2 homologous gene, and part of the AKT-type weakly inwardly rectifying channel have not been identified in the Stenotaphrum secundatum transcriptome database. A bioinformatics analysis revealed that the potassium channels of Stenotaphrum secundatum are highly conserved in terms of protein structure but have more homologous members in the same group than those of other species. Among the three species of Oryza sativa, Arabidopsis thaliana, and Setaria italica, the potassium channel of Stenotaphrum secundatum is more closely related to the potassium channel of Setaria italica, which is consistent with the taxonomic results of these species belonging to Paniceae. Subcellular location experiments demonstrate that SsKAT1.1 is a plasma membrane protein. The expression of SsKAT1.1 reversed the growth defect of the potassium absorption-deficient yeast strain R5421 under a low potassium supply, indicating that SsKAT1.1 is a functional potassium channel. The transformation of SsKAT1.1 into the cold-sensitive yeast strain INVSC1 increased the cold resistance of the yeast, indicating that SsKAT1.1 confers cold resistance. The transformation of SsKAT1.1 into the salt-sensitive yeast strain G19 increased the resistance of yeast to salt, indicating that SsKAT1.1 is involved in salt tolerance. These results suggest that the manipulation of SsKAT1.1 will improve the cold and salt stress resistance of Stenotaphrum secundatum.

Genome-Wide Identification of the Shaker Potassium Channel Family in Chinese Cabbage and Functional Studies of BrKAT1 in Yeast

Shaker potassium channels play a crucial role in potassium (K+) nutrition and stress resistance in plants. However, systematic research on Shaker K+ channels in Chinese cabbage [Brassica rapa var. chinensis (L.) Kitamura] remains scarce. This study identified 13 Shaker K+ channel members within the cabbage genome, which are unevenly distributed across eight chromosomes. Notably, the number of Shaker K+ channel members in Chinese cabbage exceeds that found in the model plants Arabidopsis (9) and rice (10). This discrepancy is attributed to a higher number of homologous proteins in Groups II and V of Chinese cabbage, with gene segmental duplication in these two subgroups being a significant factor contributing to the expansion of the Shaker K+ channel gene family. Interspecies collinearity analysis revealed that the whole genome and the Shaker K+ channel family of Chinese cabbage show greater similarity to those of Arabidopsis than to those of rice, indicating that Shaker K+ channels from the Brassicaceae family have a closer relationship than that from the Poaceae family. Given that gene expansion occurs in Group II, we investigated whether a functional difference exists between BrKAT1.1 and BrKAT1.2 using yeast assays and promoter analysis. The expression of two BrKAT1 genes in the potassium uptake-deficient yeast mutant R5421 can restore growth under low potassium conditions, indicating their role in potassium absorption. Truncation of the N-terminal 63 amino acids of BrKAT1.2 resulted in the loss of potassium absorption capability, suggesting that the N-terminus is essential for maintaining the potassium absorption function of BrKAT1.2. Furthermore, the expression of the two BrKAT1 genes in the salt-sensitive yeast G19 enhances yeast tolerance to salt stress. These results demonstrate that BrKAT1.1 and BrKAT1.2 exhibit similar abilities in potassium uptake and salt tolerance. The difference between BrKAT1.1 and BrKAT1.2 lay in their promoter regulatory elements, suggesting that differences in transcriptional regulation contributed to the functional differentiation of BrKAT1.1 and BrKAT1.2. These findings provide a foundation for understanding the evolution and functional mechanisms of the Shaker K+ channel family in Chinese cabbage and for improving potassium nutrition and salt tolerance in this species through the manipulation of BrKAT1.

The Smoky Impact of Nicotinic Acetylcholine Receptors on Testicular Function.

Smoking habits (from classic cigarettes to e-cigarettes and heated tobacco) are a relatively common finding in the medical histories of couples referred to fertility centers. Tobacco smoke and e-cigarettes may deliver many substances with known harmful effects on both general and reproductive health, including nicotine. Nicotinic Acetylcholine receptors (nAChRs) form a heterogeneous family of ion channels that are differently expressed in different tissues. According to the homomeric or heteromeric combination of at least five different subunits (named from α to ε), they have peculiar pharmacological and biophysical properties. nAChRs respond to the neurotransmitter acetylcholine, which influences a number of physiological functions not restricted to neurons and plays an important role in the structure and function of non-neuronal tissues such as the testis. nAChRs are also the target of Nicotine, the active element responsible for tobacco addiction. This review summarizes recent findings on the involvement of nAChRs in testicular physiology, highlighting the effects of nicotine exposure observed in animal studies and clinical settings. We will discuss the latest data on fertility outcomes and the implications for understanding nAChR functions in reproductive health.

Conservation of the cooling agent binding pocket within the TRPM subfamily.

Transient Receptor Potential (TRP) channels are a large and diverse family of tetrameric cation selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca 2+ . Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4 and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of TRPM4 to intracellular Ca 2+ , enhancing the sensitivity of the channel to Ca 2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related ligand binding pockets that are allosterically coupled to opening of the pore.