Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na⁺ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K⁺ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.

In voltage-gated sodium channels, a voltage-sensor domain communicates with the pore gate regions, but the details of this process remain unclear. Now the electromechanical coupling in a Na+ channel is probed by tryptophane scanning mutagenesis, together with structural modeling, with the results indicating a crucial role for the hinge regions. In a voltage-dependent sodium channel, the activation of voltage sensors upon depolarization leads to the opening of the pore gates. To elucidate the principles underlying this conformational coupling, we investigated a putative gating interface in domain III of the sodium channel using voltage-clamp fluorimetry and tryptophan-scanning mutagenesis. Most mutations have similar energetic effects on voltage-sensor activation and pore opening. However, several mutations stabilized the activated voltage sensor while concurrently destabilizing the open pore. When mapped onto a homology model of the sodium channel, most localized to hinge regions of the gating interface. Our analysis shows that these residues are involved in energetic coupling of the voltage sensor to the pore when both are in resting and when both are in activated conformations, supporting the notion that electromechanical coupling in a voltage-dependent ion channel involves the movement of rigid segments connected by elastic hinges.