Open-state structure and pore gating mechanism of the cardiac sodium channel

1. Download : Download high-res image (216KB)
2. Download : Download full-size image

     

Mutations in the pore region modify epithelial sodium channel gating by shear stress

Previous studies have shown that epithelial Na+ channels (ENaCs) are activated by laminar shear stress (LSS). ENaCs with a high intrinsic open probability because of a mutation (betaS518K) or covalent modification of an introduced Cys residue (alphaS580C) in the pre-second transmembrane domain (pre-M2) were not activated by LSS, suggesting that the pre-M2 region participates in conformational rearrangements during channel activation. We examined the role of the pore region of the alpha-subunit in channel gating by studying the kinetics of activation by LSS of wild-type ENaC and channels with Cys mutations in the tract Ser576-Ser592. Whole cell Na+ currents were monitored in oocytes expressing wild-type or mutant ENaCs prior to and following application of LSS. Following a 2.2-s delay, a monoexponential increase in Na+ currents was observed with a time constant (tau) of 8.1 s in oocytes expressing wild-type ENaC. Cys substitutions within the alpha-subunit in the tract Ser580-Ser589 resulted in: (i) a reduction (Ser580-Trp585, Gly587) or increase (Ser589) in delay times preceding channel activation by LSS, (ii) an increase (Gln581, Leu584, Trp585, Phe586, Ser588) or decrease (Ser589) in the rate of channel activation, or (iii) a decrease in the magnitude of the response (Ser583, Gly587, Leu584). Cys substitutions at a putative amiloride-binding site (alphaSer583 or betaGly525) or within the selectivity filter (alphaGly587) resulted in a reduction in the LSS response, and exhibited a multiexponential time course of activation. The corresponding gamma-subunit mutant (alphabetagammaG542C) had a minimal response to LSS and exhibited a high intrinsic open probability. These data suggest that residues in the pore region participate in the sensing and/or transduction of the mechanical stimulus that results in channel activation and are consistent with the hypothesis that the ENaC pore region has a key role in modulating channel gating.     

A mutation in the pore of the sodium channel alters gating.

Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure.     

Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.     

Asymmetric organization of the pore region of the epithelial sodium channel

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.     

Stochastic description of sodium channel gating

A stochastic model of the sodium channel is proposed. Transitions from the resting to the open state of the channel is described by the gamma distribution. The open state is temporary with an average open time T, and the channel proceeds to the inactivated state. The channel can be represented by two identical control molecules which undergo conformation transitions under changes of the electrical field. The gating of the channel is analyzed and its relation to the gating current is proposed. The movements of the control molecules are not identical with the charge movements. Charged parts of control molecules move in the electrical field of the membrane and make their conformation energetically possible. The model is represented by a set of differential equations, and explicit solutions for long depolarizing voltage steps are found. Parameters are determined to fit literary experimental data.     

The role of the putative inactivation lid in sodium channel gating current immobilization

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.     

Single point mutations of the sodium channel drastically reduce the pore permeability without preventing its gating

1. Two mutants of the sodium channel II have been expressed inXenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine.2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the openchannel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions.3. Mutant D384N has a very low permeability for any of the following ions: Cl^–, Na⁺, K⁺, Li⁺, Rb⁺, Ca²⁺, Mg²⁺, NH₄ ⁺ , TMA⁺, TEA⁺. However, asymmetric charge movements similar to the gating currents of the Na⁺-selective wild-type are still observed.4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.Abbreviations TTX tetrodotoxin; Na⁺, sodium; K⁺, potassium; - NFR normal frog Ringer - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis(-amino-ethyl ether) N,N,N',N'-tetra acetic acid - TEA tetraethylammonium - TMA tetramethylammonium;I _g , gating current; , single-channel conductance     

A physical model of sodium channel gating

Most current models of membrane ion channel gating are abstract compartmental models consisting of many undefined states connected by rate constants arbitrarily assigned to fit the known kinetics. In this paper is described a model with states that are defined in terms of physically plausible real systems which is capable of describing accurately most of the static and dynamic properties measured for the sodium channel of the squid axon. The model has two components. The Q-system consists of charges and dipoles that can move in response to an electric field applied across the membrane. It would contain and may compose the gating charge that is known to transfer prior to channel opening. The N-system consists of a charged group or dipole that is constrained to move only in the plane of the membrane and thus does not interact directly with the trans-membrane electric field but can interact electrostatically with the Q-system. The N-system has only two states, its resting state (channel closed) and its excited state (channel open) and its response time is very short in comparison with that of the Q-system. On depolarizing the membrane the the N-system will not make a transition to its open state until a critical amount of Q-charge transfer has occurred. Using only four adjustable parameters that are fully determined by fitting the equilibrium properties of the model to those of the sodium channel in the squid axon, the model is then able to describe with some accuracy the kinetics of channel opening and closing and includes the Cole and Moore delay. In addition to these predictions of the behaviour of assemblies of channels the model predicts some of the individual channel properties measured by patch clamp techniques.     

Episodic ataxia type-1 mutations in the hKv1.1 cytoplasmic pore region alter the gating properties of the channel.

Episodic ataxia type-1 is a rare human neurological syndrome which occurs during childhood and persists through the whole life of affected patients. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene hKv1.1 of different affected families. V408A and E325D mutations are located in the cytoplasmic putative pore region of hKv1.1 channels and profoundly alter their gating properties. V408A channels showed increased kinetic rates of activation, deactivation and C-type inactivation. Expression of E325D channels in Xenopus oocytes led to an approximately 13-fold current amplitude reduction and to a 52.4 mV positive shift in the voltage dependence of activation. Moreover, the E325D mutation altered the kinetics of activation, deactivation, C-type inactivation and channel open probability. Heteromeric channels composed of two wild-type and two mutated subunits, linked as dimers, showed gating properties intermediate between channels formed from four normal or four mutated subunits. The results demonstrate that the highly conserved residues Val408 and Glu325 play a pivotal role in several gating processes of a human potassium channel, and suggest a pathogenetic mechanism by which the impairment of the delayed-rectifier function of affected neurons is related to the type and number of mutated subunits which make up the hKv1.1 channels.     

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K_V and Ca_V channels. Like K_V channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca_V channels. We developed structural models of the NaChBac channel in closed and open conformations, using K⁺-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K⁺ crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K⁺ channels of known structure and Na_V, Ca_V, and TRP channels of unknown structure.     

Diverse gating in K channels: Differential role of the pore-helix glutamate in stabilizing the channel pore

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K⁺ (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71–Asp80 carboxyl–carboxylate interaction ‘behind’ the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu–Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K⁺ channels.     

A yeast genetic screen reveals a critical role for the pore helix domain in TRP channel gating

TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.     

A sodium channel gating model based on single channel, macroscopic ionic, and gating currents in the squid giant axon.

Sodium channel gating behavior was modeled with Markovian models fitted to currents from the cut-open squid giant axon in the absence of divalent cations. Optimum models were selected with maximum likelihood criteria using single-channel data, then models were refined and extended by simultaneous fitting of macroscopic ionic currents, ON and OFF gating currents, and single-channel first latency densities over a wide voltage range. Best models have five closed states before channel opening, with inactivation from at least one closed state as well as the open state. Forward activation rate constants increase with depolarization, and deactivation rate constants increase with hyperpolarization. Rates of inactivation from the open or closed states are generally slower than activation or deactivation rates and show little or no voltage dependence. Channels tend to reopen several times before inactivating. Macroscopic rates of activation and inactivation result from a combination of closed, open and inactivated state transitions. At negative potentials the time to first opening dominates the macroscopic current due to slow activation rates compared with deactivation rates: channels tend to reopen rarely, and often inactivate from closed states before they reopen. At more positive potentials, the time to first opening and burst duration together produce the macroscopic current.     

Simultaneous modifications of sodium channel gating by two scorpion toxins.

The effects of purified scorpion toxins from two different species on the kinetics of sodium currents were evaluated in amphibian myelinated nerves under voltage clamp. A toxin from Leiurus quinquestriatus slowed and prevented sodium channel inactivation, exclusively, and a toxin from Centruroides sculpturatus Ewing reduced transient sodium currents during a maintained depolarization, and induced a novel inward current that appeared following repolarization, as previously reported by Cahalan (1975, J. Physiol. [Lond.]. 244:511-534) for the crude scorpion venom. Both of these effects were observed in fibers treated with both of these toxins, and the kinetics of the induced current were modified in a way that showed that the same sodium channels were modified simultaneously by both toxins. Although the toxins can act on different sites, the time course of the action of C. sculpturatus toxin was accelerated in the presence of the L. quinquestriatus toxin, indicating some form of interaction between the two toxin binding sites.     

Ion conductance vs. pore gating and selectivity in KcsA channel: Modeling achievements and perspectives

KcsA potassium channel belongs to a wide family of allosteric proteins that switch between closed and open states conformations in response to a stimulus, and act as a regulator of cation activity in living cells. The gating mechanism and cation selectivity of such channels have been extensively studied in the literature, with a revival emphasis these latter years, due to the publication of the crystallized structure of KcsA. Despite the increasing number of research and review papers on these topics, quantitative interpretation of these processes at the atomic scale is far from achieved. On the basis of available experimental and theoretical data, and by including our recent results, we review the progresses in this field of activity and discuss the weaknesses that should be corrected. In this spirit, we partition the channel into the filter, cavity, extra and intracellular media, in order to analyze separately the specificity of each region. Special emphasis is brought to the study of an open state for the channel and to the different properties generated by the opening. The influence of water as a structural and dynamical component of the channel properties in closed and open states, as well as in the sequential motions of the cations, is analyzed using molecular dynamics simulations and ab initio calculations. The polarization and charge transfer effects on the ions’ dynamics and kinetics are discussed in terms of partial charge models.     

The quantal gating charge of sodium channel inactivation

Using a very low noise voltage clamp technique it has been possible to record from the squid giant axon a slow component of gating current (I _g ) during the inactivation phase of the macroscopic sodium current (I _Na ) which was hitherto buried in the baseline noise. In order to examine whether this slowI _g contains gating charge that originates from transitions between the open (O) and the inactivated (I) states, which would indicate a true voltage dependence of inactivation, or whether other transitions contribute charge to slowI _g , a new model independent analysis termed isochronic plot analysis has been developed. From a direct correlation ofI _g and the time derivative of the sodium conductance dg _Na/d the condition when only O-I transitions occur is detected. Then the ratio of the two signals is constant and a straight line appears in an isochronic plot ofI _g vs. dg _Na/d . Its slope does not depend on voltage or time and corresponds to the quantal gating charge of the O-I transition (q _h ) divided by the single channel ionic conductance (). This condition was found at voltages above – 10 mV up to + 40 mV and a figure of 1.21e ^– was obtained forq _h at temperatures of 5 and 15°C. At lower voltages additional charge from other transitions, e.g. closed to open, is displaced during macroscopic inactivation. This means that conventional Eyring rate analysis of the inactivation time constant _h is only valid above – 10 mV and here the figure forq _h was confirmed also from this analysis. It is further shown that most of the present controversies surrounding the voltage dependence of inactivation can be clarified. The validity of the isochronic plot analysis has been confirmed using simulated gating and ionic currents.Abbreviations I _g gating current - I _Na sodium ionic current - g _Na macroscopic sodium conductance - single channel conductance - C, O, I closed, open, inactivated state occupancy of channels - g _h quantal charge displaced in a single O-I transition of Na channel - e ^– equivalent electron charge - h index referring to inactivation process - S _l limiting slope in isochronic plot see Eq.(3) - fractional distance, see Fig. 4 and (4, 5) - TMA tetramethylammonium - TTX tetrodotoxin - Tris tris(hydroxymethyl)aminomethane - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

Intersubunit conformational changes mediate epithelial sodium channel gating

The epithelial Na⁺ channel (ENaC) functions as a pathway for Na⁺ absorption in the kidney and lung, where it is crucial for Na⁺ homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (α_K477, β_E446, and γ_E455) form interfaces with residues in adjacent subunits (β_V85, γ_V87, and α_L120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na⁺, signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na⁺ homeostasis.