Compound‐specific Na + channel pore conformational changes induced by local anaesthetics

Upon prolonged depolarizations, voltage‐dependent Na + channels open and subsequently inactivate, occupying fast and slow inactivated conformational states. Like C‐type inactivation in K + channels, slow inactivation is thought to be accompanied by rearrangement of the channel pore. Cysteine‐labelling studies have shown that lidocaine, a local anaesthetic (LA) that elicits depolarization‐dependent (‘use‐dependent’) Na + channel block, does not slow recovery from fast inactivation, but modulates the kinetics of slow inactivated states. While these observations suggest LA‐induced stabilization of slow inactivation could be partly responsible for use dependence, a more stringent test would require that slow inactivation gating track the distinct use‐dependent kinetic properties of diverse LA compounds, such as lidocaine and bupivacaine. For this purpose, we assayed the slow inactivation‐dependent accessibility of cysteines engineered into domain III, P‐segment (μ1: F1236C, K1237C) to sulfhydryl (MTSEA) modification using a high‐speed solution exchange system. As expected, we found that bupivacaine, like lidocaine, protected cysteine residues from MTSEA modification in a depolarization‐dependent manner. However, under pulse‐train conditions where bupivacaine block of Na + channels was extensive (due to ultra‐slow recovery), but lidocaine block of Na + channels was not, P‐segment cysteines were protected from MTSEA modification. Here we show that conformational changes associated with slow inactivation track the vastly different rates of recovery from use‐dependent block for bupivacaine and lidocaine. Our findings suggest that LA compounds may produce their kinetically distinct voltage‐dependent behaviour by modulating slow inactivation gating to varying degrees.