S4 Helix Research Articles

Probing Conformational Transition of TRPV5 Induced by Mechanical Force Using Coarse-Grained Molecular Dynamics.

Transient receptor potential vanilloid 5 (TRPV5) is a calcium-selective TRP channel that plays a crucial role in calcium homeostasis regulation. However, there are still many issues that need to be addressed, such as the specific conformational transition of TRPV5 and the specific functions of each structure in cation gating. Here, we build a model of the calcium ion transport protein from Xenopus oocytes in the presence of the lipid membrane and water molecules. Due to the activation process of ion channels are global and collective, coarse-grained molecular dynamics (CG-MD) simulations of the potential of mean force along the conformational transition pathway are performed. The CG-MD simulations show that the S6 helix plays a vital role in the TRPV5 conformational transition. Most importantly, these simulated trajectories indicate that the activation of ion channels happens before the extension and rotation of S6 helices, revealing that TRPV5 has a unique gating mechanism different from TRPV6. The present work demonstrates how the mechanical force acting on the S6 helix opens the TRPV5 channel gates. These results deepen our understanding of the TRPV5 gating mechanism.

Structure of human TRPM8 channel

TRPM8 is a non-selective cation channel permeable to both monovalent and divalent cations that is activated by multiple factors, such as temperature, voltage, pressure, and changes in osmolality. It is a therapeutic target for anticancer drug development, and its modulators can be utilized for several pathological conditions. Here, we present a cryo-electron microscopy structure of a human TRPM8 channel in the closed state that was solved at 2.7 Å resolution. Our structure comprises the most complete model of the N-terminal pre-melastatin homology region. We also visualized several lipids that are bound by the protein and modeled how the human channel interacts with icilin. Analyses of pore helices in available TRPM structures showed that all these structures can be grouped into different closed, desensitized and open state conformations based on the register of the pore helix S6 which positions particular amino acid residues at the channel constriction.

Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization

Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.

Inactivation of the Kv2.1 channel through electromechanical coupling.

The Kv2.1 voltage-activated potassium (Kv) channel is a prominent delayed-rectifier Kv channel in the mammalian central nervous system, where its mechanisms of activation and inactivation are critical for regulating intrinsic neuronal excitability1,2. Here we present structures of the Kv2.1 channel in a lipid environment using cryo-electron microscopy to provide a framework for exploring its functional mechanisms and how mutations causing epileptic encephalopathies3-7 alter channel activity. By studying a series of disease-causing mutations, we identified one that illuminates a hydrophobic coupling nexus near the internal end of the pore that is critical for inactivation. Both functional and structural studies reveal that inactivation in Kv2.1 results from dynamic alterations in electromechanical coupling to reposition pore-lining S6 helices and close the internal pore. Consideration of these findings along with available structures for other Kv channels, as well as voltage-activated sodium and calcium channels, suggests that related mechanisms of inactivation are conserved in voltage-activated cation channels and likely to be engaged by widely used therapeutics to achieve state-dependent regulation of channel activity.

Functional dynamics and allosteric modulation of TRPA1

The cation channel TRPA1 is a potentially important drug target, and characterization of TRPA1 functional dynamics might help guide structure-based drug design. Here, we present results from long-timescale molecular dynamics simulations of TRPA1 with an allosteric activator, allyl isothiocyanate (AITC), in which we observed spontaneous transitions from a closed, non-conducting channel conformation into an open, conducting conformation. Based on these transitions, we propose a gating mechanism in which movement of a regulatory TRP-like domain allosterically translates into pore opening in a manner reminiscent of pore opening in voltage-gated ion channels. In subsequent experiments, we found that mutations that disrupt packing of the S4-S5 linker-TRP-like domain and the S5 and S6 helices also affected channel activity. In simulations, we also observed A-967079, a known allosteric inhibitor, binding between helices S5 and S6, suggesting that A-967079 may suppress activity by stabilizing a non-conducting pore conformation-a finding consistent with our proposed gating mechanism.

Incorporating physics to overcome data scarcity in predictive modeling of protein function: A case study of BK channels.

Machine learning has played transformative roles in numerous chemical and biophysical problems such as protein folding where large amount of data exists. Nonetheless, many important problems remain challenging for data-driven machine learning approaches due to the limitation of data scarcity. One approach to overcome data scarcity is to incorporate physical principles such as through molecular modeling and simulation. Here, we focus on the big potassium (BK) channels that play important roles in cardiovascular and neural systems. Many mutants of BK channel are associated with various neurological and cardiovascular diseases, but the molecular effects are unknown. The voltage gating properties of BK channels have been characterized for 473 site-specific mutations experimentally over the last three decades; yet, these functional data by themselves remain far too sparse to derive a predictive model of BK channel voltage gating. Using physics-based modeling, we quantify the energetic effects of all single mutations on both open and closed states of the channel. Together with dynamic properties derived from atomistic simulations, these physical descriptors allow the training of random forest models that could reproduce unseen experimentally measured shifts in gating voltage, ∆V1/2, with a RMSE ~ 32 mV and correlation coefficient of R ~ 0.7. Importantly, the model appears capable of uncovering nontrivial physical principles underlying the gating of the channel, including a central role of hydrophobic gating. The model was further evaluated using four novel mutations of L235 and V236 on the S5 helix, mutations of which are predicted to have opposing effects on V1/2 and suggest a key role of S5 in mediating voltage sensor-pore coupling. The measured ∆V1/2 agree quantitatively with prediction for all four mutations, with a high correlation of R = 0.92 and RMSE = 18 mV. Therefore, the model can capture nontrivial voltage gating properties in regions where few mutations are known. The success of predictive modeling of BK voltage gating demonstrates the potential of combining physics and statistical learning for overcoming data scarcity in nontrivial protein function prediction.

A mechanistic reinterpretation of fast inactivation in voltage-gated Na+ channels

The hinged-lid model was long accepted as the canonical model for fast inactivation in Nav channels. It predicts that the hydrophobic IFM motif acts intracellularly as the gating particle that binds and occludes the pore during fast inactivation. However, the observation in recent high-resolution structures that the bound IFM motif is located far from the pore, contradicts this preconception. Here, we provide a mechanistic reinterpretation of fast inactivation based on structural analysis and ionic/gating current measurements. We demonstrate that in Nav1.4 the final inactivation gate is comprised of two hydrophobic rings at the bottom of S6 helices. These rings function in series and close downstream of IFM binding. Reducing the volume of the sidechain in both rings leads to a partially conductive, leaky inactivated state and decreases the selectivity for Na+ ion. Altogether, we present an alternative molecular framework to describe fast inactivation.

Structural basis of human Slo2.2 channel gating and modulation

The sodium-activated Slo2.2 channel is abundantly expressed in the brain, playing a critical role in regulating neuronal excitability. The Na+-binding site and the underlying mechanisms of Na+-dependent activation remain unclear. Here, we present cryoelectron microscopy (cryo-EM) structures of human Slo2.2 in closed, open, and inhibitor-bound form at resolutions of 2.6-3.2Å, revealing gating mechanisms of Slo2.2 regulation by cations and a potent inhibitor. The cytoplasmic gating ring domain of the closed Slo2.2 harbors multiple K+ and Zn2+ sites, which stabilize the channel in the closed conformation. The open Slo2.2 structure reveals at least two Na+-sensitive sites where Na+ binding induces expansion and rotation of the gating ring that opens the inner gate. Furthermore, a potent inhibitor wedges into a pocket formed by pore helix and S6 helix and blocks the pore. Together, our results provide a comprehensive structural framework for the investigation of Slo2.2 channel gating, Na+ sensation, and inhibition.

Structure of human TRPV4 in complex with GTPase RhoA

Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in thermogenesis, regulation of vascular tone, bone homeostasis, renal and pulmonary functions. TRPV4 is implicated in neuromuscular and skeletal disorders, pulmonary edema, and cancers, and represents an important drug target. The cytoskeletal remodeling GTPase RhoA has been shown to suppress TRPV4 activity. Here, we present a structure of the human TRPV4-RhoA complex that shows RhoA interaction with the membrane-facing surface of the TRPV4 ankyrin repeat domains. The contact interface reveals residues that are mutated in neuropathies, providing an insight into the disease pathogenesis. We also identify the binding sites of the TRPV4 agonist 4α-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle, and show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6. Our structures elucidate the interaction interface between hTRPV4 and RhoA, as well as residues at this interface that are involved in TRPV4 disease-causing mutations. They shed light on TRPV4 activation and inhibition and provide a template for the design of future therapeutics for treatment of TRPV4-related diseases.

Interplay between VSD, pore and membrane lipids in electromechanical coupling in HCN channels.

Hyperpolarized-activated and Cyclic Nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a 'domino effect' during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane.

Modulation of Pore Opening of Eukaryotic Sodium Channels by π-Helices in S6.

Voltage-gated sodium channels are heterotetrameric sodium selective ion channels that play a central role in electrical signaling in excitable cells. With recent advances in structural biology, structures of eukaryotic sodium channels have been captured in several distinct conformations corresponding to different functional states. The secondary structure of the pore lining S6 helices of subunits DI, DII, and DIV has been captured with both short π-helix stretches and in fully α-helical conformations. The relevance of these secondary structure elements for pore gating is not yet understood. Here, we propose that a π-helix in at least DI-S6, DIII-S6, and DIV-S6 results in a fully conductive state. On the other hand, the absence of π-helix in either DI-S6 or DIV-S6 yields a subconductance state, and its absence from both DI-S6 and DIV-S6 yields a nonconducting state. This work highlights the impact of the presence of a π-helix in the different S6 helices of an expanded pore on pore conductance, thus opening new doors toward reconstructing the entire conformational landscape along the functional cycle of Nav Channels and paving the way to the design of state-dependent modulators.

Pore residues of transient receptor potential channels canonical 1 and 4 heteromer determine channel properties.

Transient receptor potential channels canonical 1 and 4 (TRPC1 and TRPC4) are proteins belonging to the same TRPC channel family, and the two are known to form a heterotetrameric channel. TRPC4 can form a homotetrameric, nonselective cation channel by itself, but the involvement of the TRPC1 subunit changes several major characteristics of the channel. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4 as a determinant of the identity and characteristics of a heteromeric TRPC1/4 channel: decreased calcium permeability of the channel and outward-rectifying current-voltage (I-V) curve. Mutants and chimeras of the pore residues were created, and their currents were recorded using whole cell patch clamp. The lower gate mutants of TRPC4 exhibited diminished calcium permeability as measured by GCaMP6 fluorescence. Also, chimeric channels substituting the pore region of TRPC1 to TRPC4 were made to locate the pore region that is critical in the production of an outward-rectifying I-V curve characteristic of TRPC1/4 heteromeric channels.NEW & NOTEWORTHY Heteromer research has been a challenging field due to lack of structural studies. Using chimeras and single mutants, we present evidence that the pore region of TRPC1/4 heteromer contributes to determining the channel's characteristics such as calcium permeability, I-V curve, and conductance.

The membrane electric field regulates the PIP2-binding site to gate the KCNQ1 channel

Voltage-dependent ion channels underlie the propagation of action potentials and other forms of electrical activity in cells. In these proteins, voltage sensor domains (VSDs) regulate opening and closing of the pore through the displacement of their positive-charged S4 helix in response to the membrane voltage. The movement of S4 at hyperpolarizing membrane voltages in some channels is thought to directly clamp the pore shut through the S4-S5 linker helix. The KCNQ1 channel (also known as Kv7.1), which is important for heart rhythm, is regulated not only by membrane voltage but also by the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). KCNQ1 requires PIP2 to open and to couple the movement of S4 in the VSD to the pore. To understand the mechanism of this voltage regulation, we use cryogenic electron microscopy to visualize the movement of S4 in the human KCNQ1 channel in lipid membrane vesicles with a voltage difference across the membrane, i.e., an applied electric field in the membrane. Hyperpolarizing voltages displace S4 in such a manner as to sterically occlude the PIP2-binding site. Thus, in KCNQ1, the voltage sensor acts primarily as a regulator of PIP2 binding. The voltage sensors' influence on the channel's gate is indirect through the reaction sequence: voltage sensor movement → alter PIP2 ligand affinity → alter pore opening.

Structural mechanism of human oncochannel TRPV6 inhibition by the natural phytoestrogen genistein

Calcium-selective oncochannel TRPV6 is the major driver of cell proliferation in human cancers. While significant effort has been invested in the development of synthetic TRPV6 inhibitors, natural channel blockers have been largely neglected. Here we report the structure of human TRPV6 in complex with the plant-derived phytoestrogen genistein, extracted from Styphnolobium japonicum, that was shown to inhibit cell invasion and metastasis in cancer clinical trials. Despite the pharmacological value, the molecular mechanism of TRPV6 inhibition by genistein has remained enigmatic. We use cryo-EM combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to show that genistein binds in the intracellular half of the TRPV6 pore and acts as an ion channel blocker and gating modifier. Genistein binding to the open channel causes pore closure and a two-fold symmetrical conformational rearrangement in the S4–S5 and S6-TRP helix regions. The unprecedented mechanism of TRPV6 inhibition by genistein uncovers new possibilities in structure-based drug design.

The β3-subunit modulates the effect of venom peptides ProTx-II and OD1 on NaV 1.7 gating.

The voltage-gated sodium channel NaV 1.7 is involved in various pain phenotypes and is physiologically regulated by the NaV -β3-subunit. Venom toxins ProTx-II and OD1 modulate NaV 1.7 channel function and may be useful as therapeutic agents and/or research tools. Here, we use patch-clamp recordings to investigate how the β3-subunit can influence and modulate the toxin-mediated effects on NaV 1.7 function, and we propose a putative binding mode of OD1 on NaV 1.7 to rationalise its activating effects. The inhibitor ProTx-II slowed the rate of NaV 1.7 activation, whilst the activator OD1 reduced the rate of fast inactivation and accelerated recovery from inactivation. The β3-subunit partially abrogated these effects. OD1 induced a hyperpolarising shift in theV1/2 of steady-state activation, which was not observed in the presence of β3. Consequently, OD1-treated NaV 1.7 exhibited an enhanced window current compared with OD1-treated NaV 1.7-β3 complex. We identify candidate OD1 residues that are likely to prevent the upward movement of the DIV S4 helix and thus impede fast inactivation. The binding sites for each of the toxins and the predicted location of the β3-subunit on the NaV 1.7 channel are distinct. Therefore, we infer that the β3-subunit influences the interaction of toxins with NaV 1.7 via indirect allosteric mechanisms. The enhanced window current shown by OD1-treated NaV 1.7 compared withOD1-treated NaV 1.7-β3 is discussed in the context of differing cellular expressions of NaV 1.7 and the β3-subunit in dorsal root ganglion (DRG) neurons. We propose that β3, as the native binding partner for NaV 1.7 in DRG neurons, should be included during screening of molecules against NaV 1.7 in relevant analgesic discovery campaigns.

Binding of RPR260243 at the intracellular side of the hERG1 channel pore domain slows closure of the helix bundle crossing gate.

The opening and closing of voltage-dependent potassium channels is dependent on a tight coupling between movement of the voltage sensing S4 segments and the activation gate. A specific interaction between intracellular amino- and carboxyl-termini is required for the characteristically slow rate of channel closure (deactivation) of hERG1 channels. Compounds that increase hERG1 channel currents represent a novel approach for prevention of arrhythmia associated with prolonged ventricular repolarization. RPR260243 (RPR), a quinoline oxo-propyl piperidine derivative, inhibits inactivation and dramatically slows the rate of hERG1 channel deactivation. Here we report that similar to its effect on wild-type channels, RPR greatly slows the deactivation rate of hERG1 channels missing their amino-termini, or of split channels lacking a covalent link between the voltage sensor domain and the pore domain. By contrast, RPR did not slow deactivation of C-terminal truncated hERG1 channels or D540K hERG1 mutant channels activated by hyperpolarization. Together, these findings indicate that ability of RPR to slow deactivation requires an intact C-terminus, does not slow deactivation by stabilizing an interaction involving the amino-terminus or require a covalent link between the voltage sensor and pore domains. All-atom molecular dynamics simulations using the cryo-EM structure of the hERG1 channel revealed that RPR binds to a pocket located at the intracellular ends of helices S5 and S6 of a single subunit. The slowing of channel deactivation by RPR may be mediated by disruption of normal S5-S6 interactions.

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.

Dynamics transitions of the KvAP voltage sensor during lipid-dependent gating.

In response to changes in membrane potential, the S4 helix of VSD undergoes a structural transition from resting/‘Down’ to activated/‘Up’ conformation, which is an extremely important step for the functioning of a voltage-gated ion channel. It is well known that the presence of phospholipid headgroups are an absolute requirement for the normal functioning of Kv channels whereas channels reconstituted in lipids lacking the phosphate headgroups renders the channel nonfunctional. Interestingly, apart from voltage-dependent gating, the gating of KvAP can also be influenced by the change in membrane lipid composition in a voltage-independent manner (i.e., lipid-dependent gating). Here, we have performed differential lipid reconstitution using liposomes composed of phospholipids (POPC/POPG) to stabilize the activated/‘Up’ conformation and using liposomes with a mixture of phospholipids and non-phospholipids (DOPC/cholesterol) to trap the isolated KvAP-VSD in its ‘Down’ conformation. The results, using site-directed NBD fluorescence on the entire S4 helix, show that the moving part of the KvAP sensor adopts a relaxed dynamics in its ‘Down’ state whereas a restricted dynamics is observed in its ‘Up’ state. Further, a variation in hydration dynamics is observed when the S4 transitions between the two functional states, which not only reduces the relative discrete population of S4 conformations, but also alters the lifetime distribution peaks of the S4 in ‘Down’ conformation. This strongly suggests that the structural dynamics and conformational heterogeneity of the S4 is significantly altered which correlates with the change in conformation of the sensor during gating in membranes. Importantly, our ‘Down’ state results strongly indicate a bending/breakage from the middle of the otherwise straight S4-helix in ‘Up’ conformation. Overall, our results highlight the impact of altered lipid-protein interface that has a strong influence on the structural dynamics of the functionally-important conformations of the KvAP sensor.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.

The sliding helix hypothesis describes how, upon depolarization and repolarization of the membrane, the positive gating charges of the S4 helices of voltage-sensing domains (VSDs) one by one move across the membrane electrical field and thereby effect the opening and closing of ion channels. Voltage-gated calcium channels contain four VSDs, which together regulate gating of a common pore. Although these four VSDs are highly homologous, they differ from each other, e.g. in the number of gating charges and in the number and position of their countercharges. Recent atomic-resolution structures of voltage-gated calcium and sodium channels delineate the endpoints of the VSD movement upon depolarization, showing the S4 helix in the activated up-state. While the first structures of VSDs in resting states have been solved for homotetrameric (prokaryotic) channels, resting state structures of the (pseudo-)heterotetrameric eukaryotic sodium and calcium channels are still lacking. Accordingly, structural models showing the transitions from the resting to the activated state and back are still elusive. Here we addressed this problem using enhanced sampling molecular dynamics simulations of the CaV1.1 calcium channel exposed to a strong membrane potential. Indeed, starting from the known up-state, the S4 helices of the four VSDs moved down to positions closely matching the experimentally determined resting state of the bacterial NaVAb channel. Importantly, the trajectories, the transiently formed ionic interactions between gating- and countercharges, and the speed of the S4 transitions differed substantially between the four VSDs. This is consistent with data from recent mutagenesis and voltage-clamp fluorometry studies on CaV1.1 and allows relating the individual VSD movements with their putative roles in the regulation of distinct channel gating properties, as well as their role in the activation of excitation-contraction coupling in skeletal muscle.

Electrophysiological properties of TRPC1/4 heteromer determined by its pore residues.

Transient receptor potential canonical 1 (TRPC1) is widely expressed in various mammalian tissues and is involved in many physiological functions. It is known to heteromerize with TRPC4 to form a cationic channel, and it induces significant changes in channel characteristics. The huge differences in channel characteristics in the physiological environment indicates the importance of channel regulation in many tissues expressing both TRPC1 and TRPC4. In this study, we focused on the pore region (selectivity filter, pore helix, and S6 helix) of TRPC1 and TRPC4, compared their homology, and investigated how they contribute to the electrophysiological characteristics of the ion channel. We observed changes in G-V curve and I-V curve with mutations of the pore region and demonstrated that TRPC4 selectivity filter mutants lost sensitivity to G protein mediated activation. Moreover, lower gate mutants of TRPC4 exhibited diminished calcium permeability compared to wild type channels. Through chimeric channels where we substituted the pore region of TRPC1 with that of TRPC4, we were able to determine regions that are critical in producing the double-rectifying I-V curve of the homomeric TRPC4 channel.