S4-S5 Linker Research Articles

Non-Canonical Interactions between Voltage Sensors and Pore Domain in Shaker K+-Channel

W434F mutation in the pore domain (PD) of Shaker K+-channels yields non-conductive channels, useful for gating currents studies. Comparisons of Q-Vs recorded with W434F with those recorded with absence of K+ show virtually indistinguishable data. When a mutation in the S3-S4 linker, L361R, is introduced, the Q-V recorded with W434F, and the curve of K+ conductance activation by voltage (G-V) are both strongly shifted to more negative voltages, and they unexpectedly cross each other. This effect is typical when the channel has more than one open state, which does not seem to be the case according to our single channels recordings. As expected, Q-V curve recorded in the conductive Shaker containing L361R mutation does not cross its G-V. This unprecedentedly showed that W434F-containing PD can potentially affect mutant VSDs although it is virtually silent for WT VSDs. To investigate this non-canonical coupling between VSD and PD, other than via S4-S5 linker, we produced dimers of Shaker that would show a mutant VSD (L361R) CLOSE, or FAR from a mutant PD (W434F). According to the Kv1.2 crystal structure, VSDs are near the PD from the neighbor subunit and we consider that would happen similarly in dimers of shaker. VSD mutations shift the slow-inactivation curves to more negative voltages, with the effect being more intense in CLOSE compared to FAR channels. Furthermore, current peaks progressively increase at +60 mV after 100-ms pre-pulses from −180 to −100 mV differently on CLOSE and FAR channels, indicating the VSD mutation interfere with the PD inactivation according to its relative position. Our data show that VSD and PD are in close communication beyond what is predicted by the S3-S4 connection, especially when mutations are present in both domains of the protein. Support: NIH-GM030376.

Voltage Dependence and Non-Sensor Residues

The gating charges in S4 segment are highly conserved in voltage-gated K+ channels. However, a broad range of voltage dependence for charge movement and K+ conductance activation are found, suggesting these features are determined by other motifs. The extracellular S3-S4 linker shows family-related conservation. We studied the influence of residues 353-361 of that region on the voltage dependence of both gating charges movement (Q-Vs) and K+-conductance activation (G-Vs) in Shaker K+-channel. Remarkably, hydrophilic mutations in L358 and L361 produce strong shifts of both Q-Vs and G-Vs to more negative voltages. The Q-V is shifted more than −80-mV inL361R. We scanned with mutagenesis L358 (L358X) and L361 (L361X) with different amino acids (AA) and measured the mid-points of Q-V curves (Vmed) and G-V curves (V0.5). We plotted those values with several AA scales and took their coefficient of determination from a linear regression (R2). For L358X, Vmed were correlated with the residue tendency to be in a transmembrane segment (R2=0.72) and V0.5 by the hydrophobic surface area of the residue (R2=0.66). For L361X, Vmed were correlated with the residue tendency to be buried in the protein (R2=0.86) and V0.5 by the hydration potential of the residue (R2=0.66). By fitting Q-Vs to a three-state sequential model we find V0 and V1 as the voltage dependence of two simplified steps during VSD activation. The voltage sensor (VS) coupling to the pore domain (PD) was accessed by plotting V1, the last step, with V0.5 for L358X (R2=0.68) and L361X (R2=0.91). V0.5 changes in both cases are not well correlated with V0 (R2<0.45). Our data show that voltage dependence and VS-to-PD coupling can be dramatically changed by single mutations in the S3-S4 linker, and these changes cannot be explained by using well known AA hydrophobicity scales. Support:NIH-GM030376.

Biophysical Characterization of ASAP1

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. ASAP1 is based on the Gallus gallus voltage sensitive phosphatase (GgVSP) with a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Expressing this molecule in Xenopus oocytes and recording fluorescence simultaneously with gating current using the cut-open voltage clamp technique, our studies show that the gating currents of ASAP1 are significantly faster than CiVSP.

Dynamic Solvation of Protein Cavities Underlies TRPV1 Gating

TRPV1 is a member of transient receptor potential (TRP) channels family, which promotes nonselective cationic current across the membrane in response to multiple activating stimuli such as capsaicin, temperature, extracellular pH and voltage. Since TRPV1 is directly involved in nociception, it is among the primary targets for pain-relief drugs. Despite its importance, the gating mechanism of TRPV1 remains unexplored. Here, we applied a combined computational (molecular dynamics and metadynamics simulations) and experimental approach to shed light on the open-to-closed transition of this channel. In particular, we found that the gating of TRPV1 is controlled by a strictly conserved asparagine (N676) in the middle of the S6 helix: this residue is able to rotate from a conformation facing the S4-S5 linker to one facing the central pore. This rotation is correlated with the dehydration of small protein cavities between the linker and the S6 helices and hydration of the central pore. Free energy calculations confirm that the two conformational states are stable and are separated by a relatively low free energy barrier. Based on our findings, we propose a model of TRPV1 gating in which dehydration of the protein pockets determines a rotation of N676; the latter triggers water flow inside of the channel and, ultimately, the opening of the gate. Our model is in line with the known experimental data and has been tested with mutagenesis experiments.

Mixed Periodic Paralysis & Myotonia Mutant Imparts pH Sensitivity in NaV1.4

Skeletal muscle channelopathies, the majority of which are inherited as autosomal dominant mutations, are classified as either myotonia or periodic paralysis. Myotonias are defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis is associated with low potassium levels; hence it is known as hypokalemic periodic paralysis (hypoPP). Interestingly, the P1158S missense mutant located in the S4-S5 linker of the third domain of the “skeletal muscle” voltage-gated sodium channel, NaV1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. Given that intense exercise is often accompanied by blood acidosis, we decided to determine whether changes in pH would push the P1158S towards either phenotype. We previously reported that NaV1.4 is relatively insensitive to changes in extracellular pH compared to NaV1.2 and NaV1.5. The results of our experiments with P1158S indicate that the voltage-dependence of activation and steady-state fast inactivation are hyperpolarized as pH increases, and persistent currents are increased at lower pH. Thus, P1185S turns the normally pH-insensitive NaV1.4 into a proton-sensitive channel.

Divergence in Domain IV of an Electric Fish NaV Channel Tunes its Fast Inactivation to Support Rapid Firing Rates by Electro-Motorneurons

In some neuronal cell types, persistent or resurgent current through voltage-gated sodium channels enables the regular firing of “spontaneous” action potentials from a few to a hundred Hz. However, the neurons with the fastest spontaneous firing rates known are found in the nocturnal ghost knifefish (family apteronotidae), which accomplish active electrolocation via a unique neuronal electrical organ that spontaneously fires action potentials at rates exceeding 1000 Hz. Voltage-clamping of electro-motorneurons from apteronotidae revealed voltage-gated sodium currents with incomplete fast inactivation (i.e., the existence of a persistent sodium current). Unexpectedly, RT-PCR revealed that Nav 1.4b is the dominant Nav isoform in the apteronotid spinal cord, and it is expressed here nearly exclusively. We found that this gene contains five apteronotid-specific substitutions in the S4-S5 cytoplasmic linker of Domain IV, a region that has been implicated in the process of fast inactivation of mammalian voltage gated sodium channels. Using two electrode voltage clamp electrophysiology, we assayed the effects of making combinations of these substitutions in the human cardiac sodium channel Nav 1.5 (R1644W, L1647W, M1651R, I1660F, G1661S). Interestingly, when all five apteronotid substitutions are incorporated into DIV S4-5 of Nav 1.5, a persistent sodium current is observed that is qualitatively similar to that of the apterotontid Nav 1.4b in native cells. In addition, the effects observed in some partial substitutions (for example, loss of voltage dependence of inactivation in R1644W/M1651R-Nav 1.5), were apparently lost in the context of the quintuple-mutant. Taken together, these results suggest that the evolution of rapid electrical organ discharge in knifefish was driven by the tissue-specific expression of and sequence divergence within the voltage gated sodium channel Nav 1.4b. Moreover, they help define the structural requirements within the DIV S4-S5 linker for normal inactivation in mammalian sodium channels.

The clinical and genetic spectrum of catecholaminergic polymorphic ventricular tachycardia: findings from an international multicentre registry.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an ion channelopathy characterized by ventricular arrhythmia during exertion or stress. Mutations in RYR2-coded Ryanodine Receptor-2 (RyR2) and CASQ2-coded Calsequestrin-2 (CASQ2) genes underlie CPVT1 and CPVT2, respectively. However, prognostic markers are scarce. We sought to better characterize the phenotypic and genotypic spectrum of CPVT, and utilize molecular modelling to help account for clinical phenotypes. This is a Pediatric and Congenital Electrophysiology Society multicentre, retrospective cohort study of CPVT patients diagnosed at <19 years of age and their first-degree relatives. Genetic testing was undertaken in 194 of 236 subjects (82%) during 3.5 (1.4-5.3) years of follow-up. The majority (60%) had RyR2-associated CPVT1. Variant locations were predicted based on a 3D structural model of RyR2. Specific residues appear to have key structural importance, supported by an association between cardiac arrest and mutations in the intersubunit interface of the N-terminus, and the S4-S5 linker and helices S5 and S6 of the RyR2 C-terminus. In approximately one quarter of symptomatic patients, cardiac events were precipitated by only normal wakeful activities. This large, multicentre study identifies contemporary challenges related to the diagnosis and prognostication of CPVT patients. Structural modelling of RyR2 can improve our understanding severe CPVT phenotypes. Wakeful rest, rather than exertion, often precipitated life-threatening cardiac events.

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τfast = 34 ms, τslow = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2–135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the “true” mode-shift to be ∼15 mV. Interestingly, the “true” mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.

Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of 19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. 19F probes were introduced individually to S129 and L150 near the S4-S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of 19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4-S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.

The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel.

Domains in macromolecular complexes are often considered structurally and functionally conserved while energetically coupled to each other. In the modular voltage-gated ion channels the central ion-conducting pore is surrounded by four voltage sensing domains (VSDs). Here, the energetic coupling is mediated by interactions between the S4-S5 linker, covalently linking the domains, and the proximal C-terminus. In order to characterize the intrinsic gating of the voltage sensing domain in the absence of the pore domain, the Shaker Kv channel was truncated after the fourth transmembrane helix S4 (Shaker-iVSD). Shaker-iVSD showed significantly altered gating kinetics and formed a cation-selective ion channel with a strong preference for protons. Ion conduction in Shaker-iVSD developed despite identical primary sequence, indicating an allosteric influence of the pore domain. Shaker-iVSD also displays pronounced 'relaxation'. Closing of the pore correlates with entry into relaxation suggesting that the two processes are energetically related.

A competing hydrophobic tug on L596 to the membrane core unlatches S4–S5 linker elbow from TRP helix and allows TRPV4 channel to open

We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (transient receptor potential vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between the indole of W733 of the TRP helix and the backbone oxygen of L596 secures the helix/linker contact, which acts as a latch maintaining channel closure. The modeled side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model-an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Making this residue less hydrophobic (L596A/G/W/Q/K) reduces open probability [Po; loss-of-function (LOF)], likely due to altered interactions at the polar-nonpolar interface. L596I raises Po [gain-of-function (GOF)], apparently by placing its methyl group further inward and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the levels of α-carbons of H-bonded residues L596 and W733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP helix latch, respectively. These results highlight that L596 lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.

Structural Basis for Gating and Activation of RyR1

The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca(2+)) release channel required for skeletal muscle contraction. Here, we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca(2+), ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca(2+) alone induces conformational changes in the cytoplasmic assembly ("priming"), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated withgating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain.

Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.

Voltage-gated potassium (K(v)) channels are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4-S5 linker, to the potassium pore. We determined the single-particle cryo-electron microscopy structure of mammalian K(v)10.1, or Eag1, bound to the channel inhibitor calmodulin, at 3.78 angstrom resolution. Unlike previous K(v) structures, the S4-S5 linker of Eag1 is a five-residue loop and the transmembrane segments are not domain swapped, which suggest an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4-S5 linker allow calmodulin to bind to the intracellular domains and to close the potassium pore, independent of voltage-sensor position. The structure reveals an alternative gating mechanism for K(v) channels and provides a template to further understand the gating properties of Eag1 and related channels.

Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation

Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels.

Linker flexibility of IVS3-S4 loops modulates voltage-dependent activation of L-type Ca2+ channels

ABSTRACTExtracellular S3-S4 linkers of domain IV (IVS3-S4) of L-type Ca2+ channels (CaV1) are subject to alternative splicing, resulting into distinct gating profiles serving for diverse physiological roles. However, it has remained elusive what would be the determining factor of IVS3-S4 effects on CaV1 channels. In this study, we systematically compared IVS3-S4 variants from CaV1.1-1.4, and discover that the flexibility of the linker plays a prominent role in gating characteristics. Chimeric analysis and mutagenesis demonstrated that changes in half activation voltage (V1/2) or activation time constant (τ) are positively correlated with the numbers of flexible glycine residues within the linker. Moreover, antibodies that reduce IVS3-S4 flexibility negatively shifted V1/2, emerging as a new category of CaV1 enhancers. In summary, our results suggest that the flexibility or rigidity of IVS3-S4 linker underlies its modulations on CaV1 activation (V1/2 and τ), paving the way to dissect the core mechanisms and to develop innovative perturbations pertaining to voltage-sensing S4 and its vicinities.

Rational design and validation of a vanilloid-sensitive TRPV2 ion channel

Vanilloids activation of TRPV1 represents an excellent model system of ligand-gated ion channels. Recent studies using cryo-electron microcopy (cryo-EM), computational analysis, and functional quantification revealed the location of capsaicin-binding site and critical residues mediating ligand-binding and channel activation. Based on these new findings, here we have successfully introduced high-affinity binding of capsaicin and resiniferatoxin to the vanilloid-insensitive TRPV2 channel, using a rationally designed minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed TRPV2_Quad). We found that binding of resiniferatoxin activates TRPV2_Quad but the ligand-induced open state is relatively unstable, whereas binding of capsaicin to TRPV2_Quad antagonizes resiniferatoxin-induced activation likely through competition for the same binding sites. Using Rosetta-based molecular docking, we observed a common structural mechanism underlying vanilloids activation of TRPV1 and TRPV2_Quad, where the ligand serves as molecular "glue" that bridges the S4-S5 linker to the S1-S4 domain to open these channels. Our analysis revealed that capsaicin failed to activate TRPV2_Quad likely due to structural constraints preventing such bridge formation. These results not only validate our current working model for capsaicin activation of TRPV1 but also should help guide the design of drug candidate compounds for this important pain sensor.

Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels.

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3-S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3-S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3-S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3-S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3-S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant

Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca(2+), inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing single-point ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.

The S1-S2 linker determines the distinct pH sensitivity between ZmK2.1 and KAT1.

Efficient stomatal opening requires activation of KAT-type K(+) channels, which mediate K(+) influx into guard cells. Most KAT-type channels are functionally facilitated by extracellular acidification. However, despite sequence and structural homologies, the maize counterpart of Arabidopsis KAT1 (ZmK2.1) is resistant to pH activation. To understand the structural determinant that results in the differential pH activation of these counterparts, we analysed chimeric channels and channels with point mutations for ZmK2.1 and its closest Arabidopsis homologue KAT1. Exchange of the S1-S2 linkers altered the pH sensitivity between the two channels, suggesting that the S1-S2 linker is essentially involved in the pH sensitivity. The effects of D92 mutation within the linker motif together with substitution of the first half of the linker largely resemble the effects of substitution of the complete linker. Topological modelling predicts that one of the two cysteines located on the outer face section of the S5 domain may serve as a potential titratable group that interacts with the S1-S2 linker. The difference between ZmK2.1 and KAT1 is predicted to be the result of the distance of the stabilized linkers from the titratable group. In KAT1, residue K85 within the linker forms a hydrogen bond with C211 that enables the pH activation; conversely, the linker of ZmK2.1 is distantly located and thus does not interact with the equivalent titration group (C208). Thus, in addition to the known structural contributors to the proton activation of KAT channels, we have uncovered a previously unidentified component that is strongly involved in this complex proton activation network.

Dynamics and modulation studies of human voltage gated Kv1.5 channel

The voltage gated Kv1.5 channels conduct the ultrarapid delayed rectifier current (IKur) and play critical role in repolarization of action potential duration. It is the most rapidly activated channel and has very little or no inactivated states. In human cardiac cells, these channels are expressed more extensively in atrial myocytes than ventricle. From the evidences of its localization and functions, Kv1.5 has been declared a selective drug target for the treatment of atrial fibrillation (AF). In this present study, we have tried to identify the rapidly activating property of Kv1.5 and studied its mode of inhibition using molecular modeling, docking, and simulation techniques. Channel in open conformation is found to be stabilized quickly within the dipalmitoylphosphatidylcholine membrane, whereas most of the secondary structure elements were lost in closed state conformation. The obvious reason behind its ultra-rapid property is possibly due to the amino acid alteration in S4–S5 linker; the replacement of Lysine by Glutamine and vice versa. The popular published drugs as well as newly identified lead molecules were able to inhibit the Kv1.5 in a very similar pattern, mainly through the nonpolar interactions, and formed sable complexes. V512 is found as the main contributor for the interaction along with the other important residues such as V505, I508, A509, V512, P513, and V516. Furthermore, two screened novel compounds show surprisingly better inhibitory potency and can be considered for the future perspective of antiarrhythmic survey.