Omega Pore Research Articles

Omega Mutations along S4 in Nav1.2 Channels Give Insight into Domain Specific Contribution to Activation and Steady State Inactivation

We previously identified the resting state positions of the voltage sensor S4 for each domain of Nav1.2 by means of omega mutations. We found that a double gap is needed to open the omega pore (narrow part of the gating pore) resulting in detectable omega current, also known as gating pore current. At hyperpolarizing conditions, the resting state of S4 was found for double gap RR1,2QQ in domain I, II and IV and for double gap RR2,3QQ in domain III. In this work we evaluated additional conformational states of the voltage sensor S4 moving through the gating pore by further double gap mutations along S4 (second double gap 2,3QQ and third double gap 3,4QQ). Two electrode voltage clamping on X. laevis oocytes expressing rat brain sodium channels Nav1.2 was used to measure macroscopic ionic current through the alpha pore and, if present, omega current through the omega pore. In DI and DII we detected clear outward omega current for S4 mutants RK3,4QQ at depolarizing conditions. Furthermore, we found that activation of sodium currents was right shifted by about 30 mV towards higher potentials compared to the first and second double gap mutants or wild-type sodium channel. These findings suggest two sequential gating steps of S4 between resting and activated state in both domains DI and DII. In DIII and DIV no clear outward omega current could be detected at depolarized potentials either for the second or for the third double gap mutant. However steady state inactivation was strongly left shifted by about 50 to 100 mV to more hyperpolarized potentials for the second and third double gap mutant in both domains, consistent with involvement in recovery from inactivation and immobilization, respectively.

Fluorescent Visualization of Cellular Ion Fluxes

The movement of ions across biological membranes is essential for generating electrical signals, regulating ion homeostasis and cell volume, and maintaining the resting membrane potential. Currently, electrophysiology is the primary tool to detect and measure ion efflux. Although electrical recordings are extremely precise, they do not report on the spatiotemporal flux of a specific ion from the landscape of an entire cell. We have developed an optical approach to detect ion efflux from living cells by covalently attaching small-molecule, fluorescent sensors specifically to the cell's glycocalyx. Using this fluorescent sensor approach, proton efflux through voltage-gated proton channels (hHv-1) and Cl-/H+ antiporters (ClC-5) was visualized at the surface of mammalian cells. Furthermore, inward proton fluxes from a Shaker “omega pore mutant” (R362H) were also fluorescently detected, demonstrating that our approach enables the visualization of both ion accumulation and depletion at the extracellular side of the membrane. The nuts and bolts of the labeling procedure and videos of cell surface ion accumulation and depletion will be presented.

Omega pore, an alternative ion channel permeation pathway involved in the development of several channelopathies

Voltage gated ion channels (VGIC) constitute a large family of ion channels. VGIC are responsible for ions to cross the membrane. They are composed of a pore domain associated to voltage sensor domains (VSD), which regulate the function of the pore. The VSD has been recognized as the unit responsible for sensing electrical signals of all VGIC. Recently, mutations within the VSD have been studied and revealed the creation of a new permeation pathway directly through the usually non-conductive VSD. This new permeation pathway has been called omega pore or gating pore. Given the number, the diversity and the large roles of VSD, gating pores might become an important pathological defect. Indeed, several mutations have been associated to the development of several pathologies such as periodic paralysis, arrhythmias and cardiac dilatation or also the peripheral nerve hyperexcitability.

Gating pore currents, a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy

Voltage-gated ion channels (VGIC) are transmembrane proteins responsible for the generation of electrical signals in excitable cells. VGIC were first described in 1952 by Hodgkin and Huxley,1 and have since been associated with various physiological functions such as propagating nerve impulses, locomotion, and cardiac excitability. VGIC include channels specialized in the selective passage of K+, Ca2+ Na+, or H+. They are composed of 2 main structures: the pore domain (PD) and the voltage sensor domain (VSD). The PD ensures the physiological flow of ions and is typically composed of 8 transmembrane segments (TM). The VSD detects voltage variations and is composed of 4 TM (S1-S4). Given their crucial physiological role, VGIC dysfunctions are associated with diverse pathologies known as ion channelopathies. These dysfunctions usually affect the membrane expression of ion channels or voltage-dependent conformational changes of the pore. However, an increasing number of ion channelopathies, including periodic paralysis, dilated cardiomyopathy (DCM) associated with cardiac arrhythmias, and peripheral nerve hyperexcitability (PNH), have been linked to the appearance of a new pathological mechanism involving the creation of an alternative permeation pathway through the normally non-conductive VSD of VGIC. This permeation pathway is called the gating pore or omega pore.

Inactivation Voltage Sensor S4 in Domain IV of Nav1.2 Controls Immobilization of S4 in Domain III as Shown by Omega Currents

The role of S4DIV for inactivation of skeletal muscle Na channel Nav1.4 was recognized after deciphering the channelopathia Paramyotonia congenita. We showed with point mutations in the rat brain sodium channel Nav1.2 the central role of S4DIV for inactivation (Kühn and Greeff, 1999): The single mutation R4H in S4DIV slowed the recovery from inactivation about 20 times in parallel for ionic current and immobilized gating charge. Immobilization concerns about 50 % of total gating charge returning slowly to the resting state during recovery while the other half of gating charge returns very quickly. Clearly, the amount of immobilized charge is more than just the one from S4DIV. So we speculated that S4DIV would control S4s in other domains. Now, we are able to monitor the return of S4 into the resting position for each domain separately by recording the leak current of resting-state omega pore mutants (this Meeting). We find that S4DIV with the omega mutation RR12QQ shows a fast onset of omega leak current for channels at rest; however, after an inactivating prepulse, the leak current grows with the time course of recovery as expected, since this voltage sensor controls the recovery and returns into resting position accordingly. Checking the return of S4 in the other domains, we find a fast return in DI and DII while in DIII the return follows DIV. Combining these mutations with R4H in DIV, the return in both domains III and IV is about 20 times more slowly. This suggests that immobilization of gating charge across the domains is most likely achieved by the cytoplasmic loop between DIII and DIV which under control of S4DIV closes the alpha-pore and immobilizes S4DIII.

Molecular biology and biophysical properties of ion channel gating pores.

The voltage sensitive domain (VSD) is a pivotal structure of voltage-gated ion channels (VGICs) and plays an essential role in the generation of electrochemical signals by neurons, striated muscle cells, and endocrine cells. The VSD is not unique to VGICs. Recent studies have shown that a VSD regulates a phosphatase. Similarly, Hv1, a voltage-sensitive protein that lacks an apparent pore domain, is a self-contained voltage sensor that operates as an H⁺ channel. VSDs are formed by four transmembrane helices (S1-S4). The S4 helix is positively charged due to the presence of arginine and lysine residues. It is surrounded by two water crevices that extend into the membrane from both the extracellular and intracellular milieus. A hydrophobic septum disrupts communication between these water crevices thus preventing the permeation of ions. The septum is maintained by interactions between the charged residues of the S4 segment and the gating charge transfer center. Mutating the charged residue of the S4 segment allows the water crevices to communicate and generate gating pore or omega pore. Gating pore currents have been reported to underlie several neuronal and striated muscle channelopathies. Depending on which charged residue on the S4 segment is mutated, gating pores are permeant either at depolarized or hyperpolarized voltages. Gating pores are cation selective and seem to converge toward Eisenmann's first or second selectivity sequences. Most gating pores are blocked by guanidine derivatives as well as trivalent and quadrivalent cations. Gating pores can be used to study the movement of the voltage sensor and could serve as targets for novel small therapeutic molecules.

Pain enters through the side door

TRPM3 can permeate ions through two distinct pores—the central pore and a likely alternative 'omega pore'. Entry of ions through the alternative pore of TRPM3 contributes to pain generation, making it an attractive target for the design of new analgesics.

Cys Mutation + MTS Caution is Needed in Interpretation of Arg Reaction in the S4 Transmembrane Segment of a Voltage Sensing Domain (VSD) of a Voltage Gated Channel: Results of Quantum Calculations

In all previous work on voltage gated channels, mutation of the arginines on the S4 segment of the VSD, then reaction with an MTS reagent, followed by channel shutdown, has been taken to mean that the arginine was exposed on the surface from which the reagent was applied. This may require more care in interpretation. Cysteine is smaller than the arginine by about the size of the reactive sulfonate on the MTS; The mutation leaves a large cavity where the arginine side chain had been, so the MTS can reach the cysteine, possibly via the omega pore. The backbone atoms need not move. The distance between S4 and S2 or S3 remains largely unchanged. Salt bridges, (e.g., R297-E183) however, are disturbed; when cysteine is in the reactive (negative) form, it constitutes a charge reversal mutation, as the arginine was (presumably) positive. Quantum calculations on configurations of this region for R300C of the VSD of Kv1.2 show that the cys anion can fold away from the cavity where it could react, in a manner dependent on the water and protons present. See the preprint posted at http://arxiv.org/abs/1309.1373. Optimizations have been done at BLYP/6-31G∗∗ level. Acknowledgement: Computations were done at the Brookhaven National Laboratory CFN cluster, and the CUNY hpc facility.

Transient compartment-like syndrome and normokalaemic periodic paralysis due to a Cav1.1 mutation

We studied a two-generation family presenting with conditions that included progressive permanent weakness, myopathic myopathy, exercise-induced contracture before normokalaemic periodic paralysis or, if localized to the tibial anterior muscle group, transient compartment-like syndrome (painful acute oedema with neuronal compression and drop foot). 23Na and 1H magnetic resonance imaging displayed myoplasmic sodium overload, and oedema. We identified a novel familial Cav1.1 calcium channel mutation, R1242G, localized to the third positive charge of the domain IV voltage sensor. Functional expression of R1242G in the muscular dysgenesis mouse cell line GLT revealed a 28% reduced central pore inward current and a −20 mV shift of the steady-state inactivation curve. Both changes may be at least partially explained by an outward omega (gating pore) current at positive potentials. Moreover, this outward omega current of 27.5 nS/nF may cause the reduction of the overshoot by 13 mV and slowing of the upstroke of action potentials by 36% that are associated with muscle hypoexcitability (permanent weakness and myopathic myopathy). In addition to the outward omega current, we identified an inward omega pore current of 95 nS/nF at negative membrane potentials after long depolarizing pulses that shifts the R1242G residue above the omega pore constriction. A simulation reveals that the inward current might depolarize the fibre sufficiently to trigger calcium release in the absence of an action potential and therefore cause an electrically silent depolarization-induced muscle contracture. Additionally, evidence of the inward current can be found in 23Na magnetic resonance imaging-detected sodium accumulation and 1H magnetic resonance imaging-detected oedema. We hypothesize that the episodes are normokalaemic because of depolarization-induced compensatory outward potassium flux through both delayed rectifiers and omega pore. We conclude that the position of the R1242G residue before elicitation of the omega current is decisive for its conductance: if the residue is located below the gating pore as in the resting state then outward currents are observed; if the residue is above the gating pore because of depolarization, as in the inactivated state, then inward currents are observed. This study shows for the first time that functional characterization of omega pore currents is possible using a cultured cell line expressing mutant Cav1.1 channels. Likewise, it is the first calcium channel mutation for complicated normokalaemic periodic paralysis.

A Novel Cav1.1-K1245Q Mutation Leading to Hypokalemic Periodic Paralysis

Hypokalemic periodic paralysis (HypoPP) is the most common form of the periodic paralysis. It is caused by mutations in two voltage-gated ion channels of skeletal muscle, Cav1.1 (HypoPP-1) and Nav1.4 (HypoPP-2). Almost all HypoPP-causing mutations replace positive amino acids in the S4 voltage sensor of the channels. S4 mutations in Nav1.4 channels have already been shown to conduct sodium or protons through the omega pore which cause depolarization and impair the action potential generation. A leak current conducted by muscle fibers from HypoPP-1 patients was shown; however it was not formally demonstrated to be conducted by an omega pore. A novel mutation K1245Q in S4 of domain IV of Cav1.1 was identified in a patient who expressed weakness attacks with low serum potassium suggestive of HypoPP. These attacks have also been provoked by carbohydrate application. Further investigations revealed this mutation in other affected family members. To investigate possible gating alterations in the Cav1.1-K1245Q mutation, we performed whole-cell patch clamp measurements. A GLT cell line from muscular dysgenic mice (mdg) was cultured and transiently transfected with cDNA of wild type (WT) or mutant (K1245Q) Cav1.1. Calcium current amplitudes and voltage dependencies of activation of WT and K1245Q did not show significant differences in absolute or relative values. This data does not explain the patients pathogenesis. Omega currents produced by HypoPP Nav1.4 mutations might also exist in this Cav1.1-K1245Q mutant which could contribute to the phenotype of the patients. The corresponding measurements are currently performed, the results will be presented.

Pathophysiological Role of Omega Pore Current in Channelopathies

In voltage-gated cation channels, a recurrent pattern for mutations is the neutralization of positively charged residues in the voltage-sensing S4 transmembrane segments. These mutations cause dominant ion channelopathies affecting many tissues such as brain, heart, and skeletal muscle. Recent studies suggest that the pathogenesis of associated phenotypes is not limited to alterations in the gating of the ion-conducting alpha pore. Instead, aberrant so-called omega currents, facilitated by the movement of mutated S4 segments, also appear to contribute to symptoms. Surprisingly, these omega currents conduct cations with varying ion selectivity and are activated in either a hyperpolarized or depolarized voltage range. This review gives an overview of voltage sensor channelopathies in general and focuses on pathogenesis of skeletal muscle S4 disorders for which current knowledge is most advanced.

The Pore of the Voltage-Gated Proton Channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containinga VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 inthe transmembrane VSD to form the ion selectivity filter in the channel's open conformation.

Double gaps along Shaker S4 demonstrate omega currents at three different closed states

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized KvAP channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor S4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the S4-α-helix. Based on a previous finding that the mutation R1S leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2S or R3S did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in R1S mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to SS) at sequential positions (0+1, 1+2, 2+3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that S4 would swing together with S3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the S4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.

On-Off Conditions for the Omega Currents Caused by 3 Gaps Along S4 in Shaker K-Channel

We previously have shown that each replacement of a pair of long amino acid residues by short ones at 3 different sequential positions along the arginine thread of S4 in Shaker led to an omega conductance through a proteinaceous leak pore (Gamal El-Din et al., Biophys. J. 96(3) pp. 381a, 2009). For the already known omega current mutation R362S, we showed that it leaks only if paired with the native short A359. In consequence, we found that the paired arginine to serine mutations at the subsequent positions 362,365 and 365,368 also produce omega current. This was taken as evidence that S4 slides in 3 steps through a pore towards the activated state. Each pair of 2 short serines in between the thread of long arginines appears as a gap which allows a leak when in the omega pore. Here, we present further studies of the above mutants over a wider voltage range, in addition with the alpha pore open (434W) to detect the open state with alpha current. We found that S4 in all 3 omega constructs can reach the final open state where omega current has disappeared and alpha current appears. This provides clear evidence that all 3 S4 constructs - despite the arginine-to-serine gap - still proceed to the open state, although at different voltages. We also analyzed in detail the voltage range over which the omega pore remains open, i.e. is occupied by the gap. In the cases where the pore could be closed, the voltage dependence for the closing process was studied. While all constructs close in outward direction, a clear inward directed closing of the omega-pore could so far only be demonstrated for the RR/365,368/SS construct.