Voltage-dependent Na+ channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na+ channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K+ channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels.

Membrane potential regulates the activity of voltage-dependent ion channels via specialized voltage-sensing modules but the mechanisms involved in coupling voltage-sensor movement to pore opening remain unclear due to lack of resting state structures and robust methods to identify allosteric pathways. Here, using a newly developed interaction energy analysis, we probe the interfaces of the voltage-sensing and pore modules in the drosophila Shaker K+ channel. Our measurements reveal unexpectedly strong equilibrium gating interactions between contacts at the S4 and S5 helices in addition to those between S6 and S4–S5 linker. Network analysis of MD trajectories shows that the voltage-sensor and pore motions are linked by two distinct pathways- canonical one through the S4–S5 linker and a hitherto unknown pathway akin to rack and pinion coupling involving S4 and S5 helices. Our findings highlight the central role of the S5 helix in electromechanical transduction in the VGIC superfamily.

Members of the voltage-gated ion channel superfamily (VGIC) regulate ion flux and generate electrical signals in excitable cells by opening and closing pore gates. The location of the gate in voltage-gated sodium channels, a founding member of this superfamily, remains unresolved. Here we explore the chemical modification rates of introduced cysteines along the S6 helix of domain IV in an inactivation-removed background. We find that state-dependent accessibility is demarcated by an S6 hydrophobic residue; substituted cysteines above this site are not modified by charged thiol reagents when the channel is closed. These accessibilities are consistent with those inferred from open- and closed-state structures of prokaryotic sodium channels. Our findings suggest that an intracellular gate composed of a ring of hydrophobic residues is not only responsible for regulating access to the pore of sodium channels, but is also a conserved feature within canonical members of the VGIC superfamily.

Opening and closing of the central ion-conducting pore in voltage-dependent ion channels is gated by changes in membrane potential. Although a gate residue in the eukaryotic voltage-gated sodium channel has been identified, the minimal molecular determinants of this gate region remain unknown. Here, by measuring the closed- and open-state reactivity of MTSET to substituted cysteines in all the pore-lining helices, we show that the state-dependent accessibility is delineated by four hydrophobic residues at homologous positions in each domain. Introduced cysteines above these sites do not react with intracellular MTSET while the channels are closed and yet are rapidly modified while the channels are open. These findings, in conjunction with state-dependent metal cross-bridging, support the notion that the gate residues in each of the four S6 segments of the eukaryotic sodium channel form an occlusion for ions in the closed state and are splayed open on activation.

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Voltage-gated K þ (Kv) channels assemble as tetramers of a-subunits that each consist of 6 trans-membrane segments (S1-S6) and cytoplasmic Nand C-termini. The S1-S4 segments form the voltage-sensing domains while the four S5-S6 segments form the central pore. A conserved PxP motif within S6 provides flexibility to the bottom half of S6 and regulates channel gating. Subunits of the Kv5-6 and Kv8-9 subfamilies-also known as silent Kv subfamilies (KvS)-require co-assembly with Kv2 subunits to be functionally expressed. KvS subunits alter the biophysical properties of these heterotetramers compared to Kv2; e.g. Kv6.4 induces a 40 mV hyperpolarizing shift in the voltage-dependence of inactivation and potentiates the Utype inactivation. KvS subunits lack the 2nd proline of the PxP motif which has been implicated in the Kv9.3-induced effects on Kv2.1 gating. To test the effect on U-type inactivation we exchanged the Kv2.1 PIP and Kv6.4 PAT sequences. The U-type inactivation of the mutant homomeric Kv2.1(PAT) channels was strongly potentiated resulting in less than 20% of the channels in an inactivated state above þ50 mV. In addition, the voltage-dependence of activation and inactivation displayed hyperpolarizing shifts of 25 mV. Conversely, Kv6.4(PIP) subunits decreased the U-type inactivation of Kv2.1/Kv6.4(PIP) heterotetramers to a level intermediate between Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers and induced a 10 mV hyperpolarizing shift in the voltage dependence of inactivation. These results indicate that the absence of a full PxP motif contributes to Kv6.4-induced potentiation of the Kv2.1 U-type inactivation U-type inactivation. (Supported by FWO fellowships to JS and EB & grant FWO-G.0449.11N to DJS).

The gating charges in S4 segment are highly conserved in voltage-gated K þ channels. However, a broad range of voltage dependence for charge movement and K þ 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 þ-conductance activation (G-Vs) in Shaker K þ-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 À80-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 (V med) and G-V curves (V 0.5). We plotted those values with several AA scales and took their coefficient of determination from a linear regression (R 2). For L358X, V med were correlated with the residue tendency to be in a transmembrane segment (R 2 =0.72) and V 0.5 by the hydrophobic surface area of the residue (R 2 =0.66). For L361X, V med were correlated with the residue tendency to be buried in the protein (R 2 =0.86) and V 0.5 by the hydration potential of the residue (R 2 =0.66). By fitting Q-Vs to a three-state sequential model we find V 0 and V 1 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 V 1 , the last step, with V 0.5 for L358X (R 2 =0.68) and L361X (R 2 =0.91). V 0.5 changes in both cases are not well correlated with V 0 (R 2 <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.

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