TRP Domain Research Articles

Isolation, characterization and expression analysis of TRPV4 in half-smooth tongue sole Cynoglossus semilaevis

The transient receptor potential vanilloid 4 (TRPV4), another Ca2+ entry channel, belongs to the vanilloid subfamily and responds to a number of different physical and chemical stimuli and exists widely in mammals. However, our understanding of the TRPV4 in fish remains poor. Therefore, we studied the TRPV4 gene from Cynoglossus semilaevis, named CsTRPV4 that encodes a putative protein of 870 amino acids common in structure and characteristic of mammalian TRPV4, including the domains of ANK repeats, six TM, TRP domain, and CaMBD. The CsTRPV4 was expressed ubiquitously in examined tissues: higher expression in the heart, spleen, testis, and eye, but lower expression in kidney and liver. Surprisingly, the expression of CsTRPV4 in lateral line was significantly higher than in many other tissues as the CsTRPV4 was expressed significantly in the free neuromasts. In addition, CsTRPV4 in the free neuromast from the larval fish was significantly expressed in the hair cells of the free neuromasts. Therefore, the free neuromasts can act as a mechano-sensor to the mechanical stimulation in molecular level in C. semilaevis, which lays a foundation for further study of the functions of the free neuromasts.

Spinocerebellar ataxia 48 presenting with ataxia associated with cognitive, psychiatric, and extrapyramidal features: A report of two Italian families

IntroductionSpinocerebellar ataxia 48 has recently been described as an adult onset ataxia associated with a cerebellar cognitive affective syndrome, caused by a heterozygous mutation in the STUB1 gene. MethodsWe characterized the clinical and neuroimaging phenotype of eight patients from two autosomal dominant ataxia multigenerational Italian families, in whom we conducted whole exome sequencing, targeted multigene sequencing, and Sanger sequencing studies. ResultsWe describe a complex syndrome characterized by ataxia and cognitive-psychiatric disorder in all cases, variably associated with chorea, parkinsonism, dystonia, urinary symptoms, and epilepsy. MRI showed a significant cerebellar atrophy, coupled to a T2-weighted hyperintensity affecting the dentate nuclei and extending to the middle cerebellar peduncles, whereas FDG-PET studies revealed glucose hypometabolism in cerebellum, striatum, and cerebral cortex.We identified two different novel STUB1 mutations segregating in the two families. One of the two mutations, p.(Gly33Ser), occurs in the TRP domain, whereas p.(Pro228Ser) is located in the ubiquitin ligase region. DiscussionWe emphasize the similarity of the described clinical picture with that of SCAR16, an autosomal recessive ataxia caused by biallelic mutations in the same gene, and of spinocerebellar ataxia type 17, which is considered the main Huntington's disease-like syndrome. The pathogenesis of the disease and the relationship between SCA48 and SCAR16 remain to be clarified.

TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2.

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.

A multiscale model of mechanotransduction by the ankyrin chains of the NOMPC channel.

Our senses of touch and hearing are dependent on the conversion of external mechanical forces into electrical impulses by the opening of mechanosensitive channels in sensory cells. This remarkable feat involves the conversion of a macroscopic mechanical displacement into a subnanoscopic conformational change within the ion channel. The mechanosensitive channel NOMPC, responsible for hearing and touch in flies, is a homotetramer composed of four pore-forming transmembrane domains and four helical chains of 29 ankyrin repeats that extend 150 Å into the cytoplasm. Previous work has shown that the ankyrin chains behave as biological springs under extension and that tethering them to microtubules could be involved in the transmission of external forces to the NOMPC gate. Here we combine normal mode analysis (NMA), full-atom molecular dynamics simulations, and continuum mechanics to characterize the material properties of the chains under extreme compression and extension. NMA reveals that the lowest-frequency modes of motion correspond to fourfold symmetric compression/extension along the channel, and the lowest-frequency symmetric mode for the isolated channel domain involves rotations of the TRP domain, a putative gating element. Finite element modeling reveals that the ankyrin chains behave as a soft spring with a linear, effective spring constantof 22 pN/nm for deflections ≤15 Å. Force-balance analysis shows that the entire channel undergoes rigid body rotation during compression, and more importantly, each chain exerts a positive twisting moment on its respective linker helices and TRP domain. This torque is a model-independent consequence of the bundle geometry and would cause a clockwise rotation of the TRP domain when viewed from the cytoplasm. Force transmission to the channel for compressions >15 Å depends on the nature of helix-helix contact. Our work reveals that compression of the ankyrin chains imparts a rotational torque on the TRP domain, which potentially results in channel opening.

Force Transduction in the NOMPC Mechanosensitive Channel

The recent structure of the NOMPC mechanosensitive channel, responsible for hearing and touch in fly, provides a great opportunity to understand how external mechanical forces are converted into electrical impulses through the opening of ion channels in sensory cells. NOMPC is a homo-tetramer composed of a transmembrane domain containing the ion channel pore and a cytoplasmic domain made up of 4 helical chains of 150 Å length. The chains made of 29 Ankyrin repeats come into contact with each other at two points and associate with microtubules. The transmission of external forces responsible for NOMPC gating is believed to occur by tethering the Ankyrin chains to microtubules. Previous work has demonstrated that isolated Ankyrin chains behave as biological springs under extension. Here, we combine full-atom molecular dynamics simulations, normal mode analysis (NMA), and continuum mechanics to characterize the material properties of the chains under extreme compression and extension. The NMA reveals that the lowest energy modes correspond to 4-fold symmetric compression/extension along the long-axis of the channel parallel to the membrane normal, while higher energy modes correspond to symmetric displacements of the TRP domain in the channel. The finite element model reveals that the Ankyrin chains behave as a soft spring with a linear restoring force of ∼4 pN/nm for deflections ≤15 Å, and as a non-linear spring for larger deformations. Detailed force-balance analysis on the chains during compression reveal that they exert a clockwise twisting moment on the TRP domain when viewed from the cytoplasm, which is solely a consequence of the bundle geometry. This rotation of the TRP domain is consistent with the gating mechanism suggested for the closely related TRPV1 ion channel based on open and closed structure.

Structure of full-length human TRPM4

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na+-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4-S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π-π bonds and cation-π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

Functional Analysis of TRPV1 Channels with a Genetically Encoded Cross-Linker

TRP channels constitute a family of non-selective, polymodal cation channels. They can sense different nature stimulus, i.e. chemical, electric, thermic and mechanic. The members of this family are homologous to the voltage-gated cation channels superfamily. Consequently, TRP channels have a tetrameric structure, composed by four monomers with six transmembrane domains (TMD) each, and substantial N- and C-terminal intracellular domains. The S1-S4 TMD region is considered the ligand binding domain, and the pore of the channel is comprised by the S5-S6 TMD and a pore loop between them. High-resolution structures are now available from multiple members of the TRPV subfamily. These structures reveal rich interaction networks between the N- and C-terminal domains, the intracellular loops and the pore forming helixes. For instance, the TRP domain, which is formed by an extended alpha-helix from the sixth TMD, is localized in the core of this interaction network, thus serving as a putative signaling integrator between modulatory signals and the channels gating mechanism(s). In order gather site- and conformation-specific information on TRPV1 function, we applied nonsense suppression to encode the non-canonical amino acid p-Benzoylphenylalanine (BPA) into conserved aromatic sites into TRPV1 in HEK cells. This aromatic amino acid is useful for functional perturbation analysis studies and in voltage-clamp UV dependent crosslinking assays. Specific encoding of BPA was achieved with an evolved E. coli. tyrosine synthetase and tRNA. Full length channel rescue at introduced TAG sites in the presence of BPA was first demonstrated by western blot of cell lysates. Functional expression was confirmed with whole-cell voltage clamp of TRPV1 channels in HEK293 cells, demonstrating that BPA containing channels could be gated by temperature and/or capsaicin induced activation. These data set the stage for site-specific cross-linking and the potential capture of transient gating states.

Structures of the calcium-activated, non-selective cation channel TRPM4.

TRPM4 is a calcium-activated, phosphatidylinositol bisphosphate (PtdInsP2) modulated, non-selective cation channel, and belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the cryo-EM structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide binding domain (NBD) and the C-terminal coiled coil participate in the tetrameric assembly of the channel; ATP binds at NBD and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. S1-S4 domain and post-S6 TRP domain form the central gating apparatus that likely house the Ca2+ and PtdInsP2 binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and also reveal the molecular architecture of the TRPM family for the first time.

Side chain flexibility and coupling between the S4-S5 linker and the TRP domain in thermo-sensitive TRP channels: Insights from protein modeling.

The transient receptor potential (TRP) superfamily is subdivided into several subfamilies on the basis of sequence similarity, which is highly heterogeneous but shows a molecular architecture that resembles the one present in members of the Kv channel superfamily. Because of this diversity, they produce a large variety of channels with different gating and permeability properties. Elucidation of these particular features necessarily requires comparative studies based on structural and functional data. The present study aims to compilate, analyze, and determine, in a coherent way, the relationship between intrinsic side-chain flexibility and the allosteric coupling in members of the TRPV, TRPM, and TRPC families. Based on the recently determined structures of TRPV1 and TRPV2, we have generated protein models for single subunits of TRPV5, TRPM8, and TRPC5 channels. With these models, we focused our attention on the apparently crucial role of the GP dipeptide at the center of the S4-S5 linker and discussed its role in the interaction with the TRP domain, specifically with the highly-conserved Trp during this coupling. Our analysis suggests an important role of the S4-S5L flexibility in the thermosensitivity, where heat-activated channels possess rigid S4-S5 linkers, whereas cold-activated channels have flexible ones. Finally, we also present evidence of the key interaction between the conserved Trp residue of the TRP box and of several residues in the S4-S5L, importantly the central Pro. Proteins 2017; 85:630-646. © 2016 Wiley Periodicals, Inc.

Intra-Molecular Connectivity in Sensory Receptors of the TRP Family

Membrane voltage, ligand binding, mechanical force and temperature can all induce conformational changes that open ion channel pores. A key question in understanding ion channel function is how the protein domains involved in sensing stimuli communicate with the pore to modulate the state of ion channel's gate. Transient Receptor Potential (TRP) proteins are a large and diverse family of ion channels. Although great advances have been made regarding the activation and modulation of TRP channel activity, detailed molecular mechanisms governing TRP channel gating are still needed. Temperature-activated TRP channels are tightly associated with the detection and integration of sensory input from multiple sensory modalities, emerging as a model to study the inner-workings of polymodal activation. In this context, the intracellular helix dubbed TRP domain (TD) has been suggested as an integrator of input stimuli in TRP channels, however, the mechanics of such integration is currently unknown. Here we show an extensive bioinformatic analysis to fish for bona fide TRP channels from unicellular eukaryotes and compare their primary sequence with orthologs from higher organisms. Evolutionary coupled analysis highlights important interactions connecting the TD with other portions of the channel. Finally, our predictions in combination with genetically encoded non-natural amino acids allow us to trap the open conformation of TRPV1 channels. We propose a mechanistic model for TRP domain-associated integration of the input signals.

Cryo-electron microscopy structure of the TRPV2 ion channel.

Transient receptor potential vanilloid (TRPV) cation channels are polymodal sensors involved in a variety of physiological processes. TRPV2, a member of the TRPV family, is regulated by temperature, by ligands, such as probenecid and cannabinoids, and by lipids. TRPV2 has been implicated in many biological functions, including somatosensation, osmosensation and innate immunity. Here we present the atomic model of rabbit TRPV2 in its putative desensitized state, as determined by cryo-EM at a nominal resolution of ~4 Å. In the TRPV2 structure, the transmembrane segment 6 (S6), which is involved in gate opening, adopts a conformation different from the one observed in TRPV1. Structural comparisons of TRPV1 and TRPV2 indicate that a rotation of the ankyrin-repeat domain is coupled to pore opening via the TRP domain, and this pore opening can be modulated by rearrangements in the secondary structure of S6.

Novel Alleles of gon-2, a C. elegans Ortholog of Mammalian TRPM6 and TRPM7, Obtained by Genetic Reversion Screens.

TRP (Transient Receptor Potential) cation channels of the TRPM subfamily have been found to be critically important for the regulation of Mg2+ homeostasis in both protostomes (e.g., the nematode, C. elegans, and the insect, D. melanogaster) and deuterostomes (e.g., humans). Although significant progress has been made toward understanding how the activities of these channels are regulated, there are still major gaps in our understanding of the potential regulatory roles of extensive, evolutionarily conserved, regions of these proteins. The C. elegans genes, gon-2, gtl-1 and gtl-2, encode paralogous TRP cation channel proteins that are similar in sequence and function to human TRPM6 and TRPM7. We isolated fourteen revertants of the missense mutant, gon-2(q338), and these mutations affect nine different residues within GON-2. Since eight of the nine affected residues are situated within regions that have high similarity to human TRPM1,3,6 and 7, these mutations identify sections of these channels that are potentially critical for channel regulation. We also isolated a single mutant allele of gon-2 during a screen for revertants of the Mg2+-hypersensitive phenotype of gtl-2(-) mutants. This allele of gon-2 converts a serine to phenylalanine within the highly conserved TRP domain, and is antimorphic against both gon-2(+) and gtl-1(+). Interestingly, others have reported that mutation of the corresponding residue in TRPM7 to glutamate results in deregulated channel activity.

The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating

Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.

Substrate specificity and biochemical characterization of an adenylation domain from an obligate marine actinomycete

Salinispora arenicola CNS-205 was a first-isolated obligate marine actinomycete. A gene (sare0357), annotated as ''amino acid adenylation domain'' located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357 was expressed in E. coli. Sare0357 specifically recognized and activated tryptophan (Trp) and phenylalanine (Phe). The basic kinetic parameters of Sare0357 for Trp were K m = 0.04 mM, V max = 2.1 μM/min, k cat = 14.2 min(-1), and for Phe were K m = 0.03 mM, V max = 1.6 μM/min, kcat = 10.4 min(-1). Our data elucidated Sare0357 biological role and biochemical properties as a Trp and Phe-activating adenylation domain.

The L596-W733 Bond between S4-S5 Linker and TRP Domain Maintains Basal Activity and Enables Inactivation of TRPV4

Unlike other cation channels, all TRP (Transient Receptor Potential) channel subunits have a TRP-domain helix immediately trailing S6 that bears the gate. The role(s) of TRP-domain helix is unclear. Recent cryo-EM TRPV1 structures revealed that this helix forms a bond with the beginning of the S4-S5 linker. By homology modeling, we identified the corresponding L596-W733 bond in TRPV4 (Vanilloid type 4). L596P, likely a gain-of-function (GOF) mutation, causes bone-developmental blockage of the spondylometaphyseal dysplasia Kozlowski type (SMDK) in human. Our previous screen also isolated W733R as a GOF that suppresses grows of yeast expressing TRPV4. Here we show, when expressed in Xenopus oocytes, TRPV4 with L596P or W733R mutation displays normal depolarization-induced activation and outward rectification. However, as expected from their biological GOF phenotypes, these mutant channels indeed have higher basal open probabilities and limited responses to the strong agonist GSK1016790A. In addition, W733R current also fails to inactivate after activation during depolarization. Systematic substitutions of W733 with amino acids of different properties produce similar electrophysiological defects. The results can be consistently interpreted in the context of the homology model of TRPV4 that we have developed. Our results indicate that the TRP domain stabilizes various functional conformations by bonding to other structures, especially to the S4-S5 linker.

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P2 interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.

The Molecular Determinants of PI(4,5)P2 Binding to TRPV1 Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site PI(4,5)P2 interacts with the amino acid residues R575 and R579 in the S4-S5 linker and with K694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that R575, R579 and K694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which leads to the opening of the lower TRPV1 channel gate.

A Single-Residue Switch for High Temperature Dependence of Thermal TRPV3 Channels

Thermal TRP channels, a group of ion channels from the transient receptor potential family, play important functions in pain transduction and thermal sensation. The channels are directly activated by temperature, with strong temperature dependence. However, how temperature gates these channels remains poorly understood. One characteristic feature of some thermal TRP channels is hysteresis of gating. This is perhaps most prominent in vanilloid receptor TRPV3. The channel is initially only responsive to extreme heat with low activity, and only after repeated stimulation it becomes vigorously responsive to warm temperatures. Importantly, the hysteresis of activation is accompanied with a profound loss of the high temperature dependence of the channel, suggesting that the hysteresis involves structural changes in temperature sensing. Elucidation of such changes will therefore provide an opportunity to unravel the molecular mechanism underlying temperature sensing of thermal channels. Here we examine the molecular basis underlying hysteresis of heat activation of TRPV3. We show that the hysteresis is mediated by an N terminal domain proximal to membranes, the same region that we have previously identified for temperature sensing in heat sensitive vanilloid receptors. Upon exchange of the region, the heat activation of the channel becomes reversible and the temperature dependence becomes considerably reduced as the wild type channels after sensitization. Interesting much of the hysteresis effect can be attributed to a single residue near the TRP box. The position of the residue suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain.

Negatively Charged Amino Acids Near and in Transient Receptor Potential (TRP) Domain of TRPM4 Channel Are One Determinant of Its Ca2+ Sensitivity

Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+).

Dissecting domain-specific evolutionary pressure profiles of transient receptor potential vanilloid subfamily members 1 to 4.

The transient receptor potential vanilloid family includes four ion channels–TRPV1, TRPV2, TRPV3 and TRPV4–that are represented within the vertebrate subphylum and involved in several sensory and physiological processes. These channels are related to adaptation to the environment, and probably under strong evolutionary pressure. Using multiple sequence alignments as source for evolutionary, bioinformatics and statistical analysis, we have analyzed the evolutionary profiles for TRPV1, TRPV2, TRPV3 and TRPV4. The evolutionary pressure exerted over vertebrate TRPV2 sequences compared to the other channels argues for a positive selection profile for TRPV2 compared to TRPV1, TRPV3 and TRPV4. We have analyzed the selective pressure on specific protein domains, observing a common selective pressure trend for the common TRPV scaffold, consisting of the ankyrin repeat domain, the membrane proximal domain, the transmembrane domain, and the TRP domain. Through a more detailed analysis we have identified evolutionary constraints involved in the subunit contact at the transmembrane domain level. Performing evolutionary comparison, we have translated specific channel structural information such as the transmembrane topology, and the interaction between the membrane proximal domain and the TRP box. We have also identified potential common regulatory domains among all TRPV1-4 members, such as protein-protein, lipid-protein and vesicle trafficking domains.