Pore Turret Research Articles

Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy.

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

A mutational and modeling study of a binding pocket for IKs activators between KCNQ1 and KCNE1.

The IKs current formed by the co-assembly of KCNQ1 and KCNE1 plays an important role in cardiac repolarization. Loss-of-function mutations in KCNQ1 and KCNE1 are linked to LQT syndrome. Mefenamic acid (Mef) an NSAID, and DIDS a stilbene, are known to enhance IKs currents. The present study explores in detail the role of previously identified K41-KCNE1 in Mef action as well as uncovering the KCNQ1/KCNE1 binding site(s) of Mef and DIDS. In silico experiments of drug docking on a model of IKs channel and molecular dynamic (MD) simulations were used to map critical KCNQ1 and KCNE1 residues, followed by site-directed mutagenesis and functional analysis using whole cell patch clamp. Docking procedures and MD simulations show that Mef and DIDS bind between extracellular KCNE1 residues, specifically to the subunits of the external S6 helix; neighbouring S1; and the pore turret of the opposing subunit. Mef transforms the slowly activating WT IKs current (4:4 EQ) into a current with an almost linear waveform, which causes a left shift in activation V1/2 and inhibits tail current decay at −40 mV. These effects are prevented by the K41C-KCNE1 mutation in a stoichiometric manner. MD simulations and mutagenesis scanning identified three critical residues for Mef binding: W323-KCNQ1; Y46-KCNE1; and K41-KCNE1 in which Mef effects are greatly diminished. The effect of DIDS on WT-EQ are less pronounced than Mef, and K41C-KCNE1 was unable to fully prevent effects of DIDS. Our results show that both activators bind in the same general channel region but depend on different critical residues. These findings provide insights into a common drug-induced binding site, suggesting that the chemical structure of either compound could serve as a starting point for the development of IKs agonists.

A specialized pore turret in the mammalian cation channel TRPV1 is responsible for distinct and species-specific heat activation thresholds

The transient receptor potential vanilloid 1 (TRPV1) channel is a heat-activated cation channel that plays a crucial role in ambient temperature detection and thermal homeostasis. Although several structural features of TRPV1 have been shown to be involved in heat-induced activation of the gating process, the physiological significance of only a few of these key elements has been evaluated in an evolutionary context. Here, using transient expression in HEK293 cells, electrophysiological recordings, and molecular modeling, we show that the pore turret contains both structural and functional determinants that set the heat activation thresholds of distinct TRPV1 orthologs in mammals whose body temperatures fluctuate widely. We found that TRPV1 from the bat Carollia brevicauda exhibits a lower threshold temperature of channel activation than does its human ortholog and three bat-specific amino acid substitutions located in the pore turret are sufficient to determine this threshold temperature. Furthermore, the structure of the TRPV1 pore turret appears to be of physiological and evolutionary significance for differentiating the heat-activated threshold among species-specific TRPV1 orthologs. These findings support a role for the TRPV1 pore turret in tuning the heat-activated threshold, and they suggest that its evolution was driven by adaption to specific physiological traits among mammals exposed to variable temperatures.

Identification of a proton sensor that regulates conductance and open time of single hERG channels

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.

Structures of TRPV2 in distinct conformations provide insight into role of the pore turret.

Cation channels of the TRP family serve important physiological roles by opening in response to diverse intra-and extra-cellular stimuli which regulate their lower or upper gates. Despite extensive studies, the mechanism coupling these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as “pore turrets,” which are proximal to the upper gates. We establish the importance of the pore turret through activity assays and by solving structures of rat TRPV2 both with and without an intact turret at resolutions of 4.0 Å and 3.6 Å respectively. These structures resolve the full-length pore turret and reveal fully open and partially open states of TRPV2, both with unoccupied vanilloid pockets. Our results suggest a mechanism by which physiological signals, such as lipid binding, can regulate the lower gate and couple to the upper gate through a pore turret-facilitated mechanism.

TRPV1 pore turret dictates distinct DkTx and capsaicin gating

Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans, DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel's outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation.

Molecular Mechanism of TRPV2 Channel Pore Dynamics during Ligand Activation

The transient receptor potential (TRP) superfamily, one of the largest families of the non-selective cation channels, is subdivided into six major branches: TRPV, TRPC, TRPM, TRPA, TRPP, and TRPML. The TRPV subfamily contains six unique members, four non-selective cation channels (TRPV1-TRPV4) and two highly Ca2+ selective channels (TRPV5 and TRPV6). A hallmark of these polymodal TRP channels is their exceptionally high permeability to large organic cations in response to prolonged ligand stimulation. The pore dilation phenomenon has been proposed to play a role in this process. Although cryo-electron microscopy structures of various TRP channels have been resolved in both apo and ligand bound states, the structural basis of this uptake is unknown due to unavailability of the full-length TRP structures in ligand-activated states. Recent cryo-electron microscopy (cryo-EM) structure of full-length rat TRPV2 in the apo state revealed details about TRPV2 channel architecture, but did not elucidate aspects of channel activation and gating. We have determined a ligand-activated TRPV2 structure, which displayed a distinct channel gating mechanism that involves rearrangement of the S5 helix, pore turret and selectivity filter domains. Together these results provide foundation to further understanding TRP channels large organic cations’ permeation, their gating properties and divergent physiological functions.

TRPV2 Ion Channel Gating Through Allosteric Domain Coupling Revealed by Cryo-EM

Cation channels of the TRP family serve important physiological roles by opening in response to diverse intra- and extra-cellular stimuli which regulate their lower or upper gates. Despite extensive studies, the mechanism which couples these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as the “pore turret,” which are proximal to the upper gate domains. We establish the importance of the pore turret through activity assays and by solving structures of TRPV2 both with and without this domain intact at resolutions of 4.0 A and 3.6 A respectively. These structures resolve the full length pore turret and reveal fully open and closed states of TRPV2 with an unoccupied vanilloid pocket. Our results reveal how physiological signals, such as PIP2 binding, can regulate the lower gate and couple to the upper gate through a mechanism facilitated by the pore turret.

Amino Acid Properties of Trafficking Determinants in the Outer Pore-Forming Region of Kv1 Potassium Channels in Cell Lines

Different classes of Kv1 potassium channels have different trafficking patterns despite having very similar amino acid sequences. Two amino acids responsible for these differences have been identified in the outer pore turret region of Kv1.1 and Kv1.4. Here we tested a series of substitutions at these two determinants on Kv1.4. All P506 substitutions tested resulted in a significant decrease in surface protein, total protein, and protein half-life, indicating that proline is required at 506 to stabilize protein conformation and increase trafficking to the cell surface. All K533 substitutions tested had no effect on total protein, suggesting that the lysine at 533 is not important for maintaining Kv1.4 protein conformation. However, a basic or long polar amino acid, such as K, R, or Q, at this position favored high surface protein and efficient trafficking of Kv1.4, whereas an acidic or short amino acid, such as D, E, S, L, N, or H, at this position induced partial high endoplasmic reticulum-retention. This intracellular retention was not due to protein misfolding. We propose that these four prolines and four lysines in a Kv1.4 homotetramer might provide a binding site for a putative endoplasmic reticulum-export molecule to ensure high cell surface protein expression of the channel.

Role of the Outer Pore Domain in Transient Receptor Potential Vanilloid 1 Dynamic Permeability to Large Cations

Transient receptor potential vanilloid 1 (TRPV1) has been shown to alter its ionic selectivity profile in a time- and agonist-dependent manner. One hallmark of this dynamic process is an increased permeability to large cations such as N-methyl-D-glucamine (NMDG). In this study, we mutated residues throughout the TRPV1 pore domain to identify loci that contribute to dynamic large cation permeability. Using resiniferatoxin (RTX) as the agonist, we identified multiple gain-of-function substitutions within the TRPV1 pore turret (N628P and S629A), pore helix (F638A), and selectivity filter (M644A) domains. In all of these mutants, maximum NMDG permeability was substantially greater than that recorded in wild type TRPV1, despite similar or even reduced sodium current density. Two additional mutants, located in the pore turret (G618W) and selectivity filter (M644I), resulted in significantly reduced maximum NMDG permeability. M644A and M644I also showed increased and decreased minimum NMDG permeability, respectively. The phenotypes of this panel of mutants were confirmed by imaging the RTX-evoked uptake of the large cationic fluorescent dye YO-PRO1. Whereas none of the mutations selectively altered capsaicin-induced changes in NMDG permeability, the loss-of-function phenotypes seen with RTX stimulation of G618W and M644I were recapitulated in the capsaicin-evoked YO-PRO1 uptake assay. Curiously, the M644A substitution resulted in a loss, rather than a gain, in capsaicin-evoked YO-PRO1 uptake. Modeling of our mutations onto the recently determined TRPV1 structure revealed several plausible mechanisms for the phenotypes observed. We conclude that side chain interactions at a few specific loci within the TRPV1 pore contribute to the dynamic process of ionic selectivity.

Divalent cations activate TRPV1 through promoting conformational change of the extracellular region

Divalent cations Mg2+ and Ba2+ selectively and directly potentiate transient receptor potential vanilloid type 1 heat activation by lowering the activation threshold into the room temperature range. We found that Mg2+ potentiates channel activation only from the extracellular side; on the intracellular side, Mg2+ inhibits channel current. By dividing the extracellularly accessible region of the channel protein into small segments and perturbing the structure of each segment with sequence replacement mutations, we observed that the S1–S2 linker, the S3–S4 linker, and the pore turret are all required for Mg2+ potentiation. Sequence replacements at these regions substantially reduced or eliminated Mg2+-induced activation at room temperature while sparing capsaicin activation. Heat activation was affected by many, but not all, of these structural alternations. These observations indicate that extracellular linkers and the turret may interact with each other. Site-directed fluorescence resonance energy transfer measurements further revealed that, like heat, Mg2+ also induces structural changes in the pore turret. Interestingly, turret movement induced by Mg2+ precedes channel activation, suggesting that Mg2+-induced conformational change in the extracellular region most likely serves as the cause of channel activation instead of a coincidental or accommodating structural adjustment.

Cyclic Nucleotide-Gated Channel Subunit Glycosylation Regulates Matrix Metalloproteinase-Dependent Changes in Channel Gating

Cyclic-nucleotide gated (CNG) channels are essential for phototransduction within retinal photoreceptors. We have demonstrated previously that the enzymatic activity of matrix metalloproteinase-2 and -9, members of the matrix metalloproteinase (MMP) family of extracellular, Ca(2+)- and Zn(2+)-dependent proteases, enhances the ligand sensitivity of both rod (CNGA1 and CNGB1) and cone (CNGA3 and CNGB3) CNG channels. Additionally, we have observed a decrease in the maximal CNG channel current (Imax) that begins late during MMP-directed gating changes. Here we demonstrate that CNG channels become nonconductive after prolonged MMP exposure. Concurrent with the loss of conductive channels is the increased relative contribution of channels exhibiting nonmodified gating properties, suggesting the presence of a subpopulation of channels that are protected from MMP-induced gating effects. CNGA subunits are known to possess one extracellular core glycosylation site, located at one of two possible positions within the turret loop near the pore-forming region. Our results indicate that CNGA glycosylation can impede MMP-dependent modification of CNG channels. Furthermore, the relative position of the glycosylation site within the pore turret influences the extent of MMP-dependent proteolysis. Glycosylation at the site found in CNGA3 subunits was found to be protective, while glycosylation at the bovine CNGA1 site was not. Relocating the glycosylation site in CNGA1 to the position found in CNGA3 recapitulated CNGA3-like protection from MMP-dependent processing. Taken together, these data indicate that CNGA glycosylation may protect CNG channels from MMP-dependent proteolysis, consistent with MMP modification of channel function having a requirement for physical access to the extracellular face of the channel.

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate.

Matrix Metalloproteinase-9 Increases the Ligand Sensitivity of CNG Channels: Evidence for Sequential Subunit Modification

Matrix metalloproteinases (MMPs) are a family of over 20 cell-surface and secreted, Zn2+- and Ca2+-dependent endopeptidases. Two closely related gelatinase MMPs, −9 and −2, have been implicated in both normal retinal processes and in several retinal diseases. MMPs are classically associated with tissue remodeling, but have been shown to influence ion channel function—either indirectly (e.g., through modification of the pericellular environment) or directly (e.g., by proteolysis of channel subunits). Because MMPs have been localized within the interphotoreceptor matrix, we hypothesized that MMP catalytic activity can influence the function of photoreceptor cyclic nucleotide-gated (CNG) channels. Cone and rod CNG channels were individually expressed in Xenopus oocytes and examined via patch-clamp recording in the inside-out configuration. Extracellular exposure to MMP-9 or −2 produced a profound increase in the efficacy of cAMP and the apparent cGMP affinity for both homomeric (CNGA3, K1/2,GMP, mean ± S.E.M: MMP960min = 0.62 μM ± 0.21; control60min = 12.6 μM ± 1.7) and heteromeric (CNGA3+CNGB3, K1/2,GMP: MMP980min = 2.2 μM ± 0.6; control80min = 16.4 μM ± 1.2) channel compositions. The gating changes required MMP-catalytic activity, exhibited complex state-dependence and were attenuated by N-glycosylation in the pore turret of CNGA subunits. Furthermore, MMP exposure resulted in decreased abundance of the full length CNGA3 subunit on immunoblots, and a concomitant increase in putative degradation products, consistent with proteolysis of core-channel subunits. Analyses of cGMP dose-response curves and current fluctuations suggest that MMPs generate channel subpopulations by sequentially modifying individual CNG channel subunits, where the apparent affinity for cGMP is governed by the total number of modified subunits (from 0 to 4). Together, these results provide evidence that MMPs can incrementally regulate the ligand sensitivity of CNG channels by proteolytic modification of CNGA subunits.

The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Functional characterization of the LQT2-causing mutation R582C and the associated voltage-dependent fluorescence signal

The R582C mutation is one of many Long-QT Syndrome type 2 (LQT2)-causing mutations localized to the human ether-a-go-go related gene (hERG) channel's S5-P linker subdomain, yet its specific mechanism of dysfunction has not been examined. This study sought to characterize the biophysical properties of the congenital LQT2-causing mutation, R582C, and utilize this mutation to provide the first report of voltage-dependent fluorescence from the S5-P linker. Properties of the R582C channels were characterized by heterologous expression in both HEK293 cells and Xenopus oocytes using a combination of patch-clamp, 2-electrode voltage-clamp, immunoblot assay, and voltage-clamp fluorimetry. Expression of hERG R582C was found to be deficient in HEK293 cells, yet was amenable to rescue by incubation at reduced temperature or by treatment with dofetilide. Rescued channels expressed at levels comparable to wild type (WT) channels. Kinetic differences result in decreased outward repolarizing current evoked by an action potential clamp protocol. Voltage-clamp fluorimetry experiments utilized the introduced cysteine to covalently attach a fluorescent probe (tetramethylrhodamine-5-maleimide) to the S5-P linker to directly observe conformational changes occurring due to inactivation. The major mechanism underlying pathogenicity of the R582C mutation is a trafficking deficiency, although channels also exhibit kinetic deficiencies, perhaps reflecting the position of the mutation in the pore turret. Voltage clamp fluorescence signals from R582C channels provide evidence that the hERG turret undergoes distinct conformational changes during inactivation.

The Pore Turret of ThermoTRP Channels is a Portable Domain Contributing to Temperature Sensing

ThermoTRP channels sense ambient temperature and function as thermosensor, however, the mechanism for sensitive temperature-dependent activation remains unknown. Recent studies point to the outer pore region as an important gating structure. Our previous study shows pore turret moves substantially during temperature-driven activation, which isn’t seen during capsaicin-driven activation. Present research focuses on contribution of pore turret to heat-induced activation using a structural perturbation approach. Partial deletions and sequence replacements are performed with TRPV1 turret. Additionally, the turret is swamped between TRPV1 and TRPV2/TRPV3. All mutant channels exhibit similar capsaicin sensitivity to wildtype TRPV1 with a less than 10-fold shift in EC50, which is consistent with the notion that temperature and ligand activate TRPV1 through different pathways. In contrast, deletion and replacement mutants exhibit activation at a very different temperature, show rapid inactivation during heating, or lose temperature response. Interestingly, TRPV1 chimera containing TRPV3 turret V1/3S behaves like wildtype TRPV3 and activates at a much lower temperature, while TRPV1 chimera containing TRPV2 turret V1/2S activates at a much higher temperature like wildtype TRPV2. These results reveal that pore turret is a portable domain contributing to temperature sensing.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Extracellular Cation Gates TRPV1 via the Heat Activation Pathway

ThermoTRP channels are expressed in sensory neurons where they detect a variety of physical and chemical stimuli including temperature, ionic strength, membrane voltage and ligand binding. However, the precise mechanisms underlying such diverse modes of activation remain largely unknown. It is found that temperature and capsaicin activate TRPV1 through separate pathways. Here we investigate how extracellular cation activates the heat-sensitive TRPV1 channel.At room temperature, Mg++ ions effectively and selectively activate TRPV1 from the extracellular side in a dose-dependent manner. The relative open probability induced by 100 mM [Mg++]out is approximately 40% of that elicited by saturating capsaicin, while the same concentration of [Mg++]in is ineffective. Mg++ substantially shifts the temperature sensitivity of the channel. At 10 mM, Mg++ shifts the takeoff temperature of TRPV1 greatly, which implies that the effects of heat and Mg++ are coupled tightly. Furthermore, mutant channels with an artificial pore turret, which have less temperature sensitivity but maintain normal capsaicin responses, also show reduced cation responses. In summary, our observations suggest that in TRPV1 the pore turret may detect both heat and ion strength, and effectively transfer the energy to open the activation gate.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Temperature Dependence of Mutant Vanilloid Receptor (tRPV) Channels

Temperature-gated transient receptor potential (TRP) channels exhibit exceedingly large energetic changes in activation. However, the source of energy and its structural basis are not known. Several studies have recently suggested that the pore domain of the channels plays an essential role in temperature gating. In particular, multiple mutations in the outer pore of the TRPV3 channel, which is activated by heat above 30oC, were found to selectively abrogate the heat response of the channel while leaving the response to chemical agonists largely intact. Vanilloid receptors (TRPV) have a membrane topology resembling that of voltage-gated ion channels, and these mutant residues are located at the S6-linker region. The profound and yet selective effects of the region on thermal sensitivity of TRPV3 suggest that the outer pore of the channel may be responsible for heat sensing. In support of the hypothesis, the replacement of a pore turret region in TRPV1 by a glycine-based fragment was also shown to specifically eliminate the heat sensitivity of the channel without compromising its capsaicin response. Alternatively, the “heat sensors” of the channels, if any, may reside elsewhere, while the pore domain is involved in the downsteam pathway of activation such as allosteric coupling between the heat sensors and the gate of the channel. In this study, we will attempt to discern such two mechanisms. We will show the remaining of temperature dependence in the mutant channels and explore mechanisms and the role of the pore domain in temperature gating.

Reply to Yao et al.: Is the pore turret just thermoTRP channels’ appendix?

Numerous studies of thermosensitive transient receptor potential (thermoTRP) channels have focused on the outer pore that contains sites critical for the channel's sensitivity to temperature, acidification, spider toxin, and other gating modulators (Fig. 1). Two segments of each subunit contribute to the outer pore structure: one is between S5 and the pore helix, the other is between the selectivity filter and S6. For the convenience of discussion, we call them the large turret and the small turret, respectively. From the available structural information of tetrameric channels, it is expected that the two turrets interact extensively. Indeed, the sites affecting gating spread across the outer pore landscape. Of particular interests are a number of sites that when mutated, lead to permanent opening or closing of the temperature-activation gate (1–3).