Mechanosensitivity of a Voltage-Gated Ion Channel, Nav1.5

Abstract

Voltage-gated ion channels are often found in tissues where electrical and mechanical stimuli coexist. The mechanosensitive, voltage-gated sodium channel Nav1.5 (encoded by SCN5A) is expressed in two such electromechanical organs, the heart and the gastrointestinal tract. Mutations in SCN5A are frequently pathogenic and may affect mechanoelectrical coupling. The aim of this study was to assess mechanical sensitivity of Nav1.5 at the molecular level. SCN5A was expressed in HEK cells and studied using a pipette pulled and fire polished to ensure that a small number (2-50) of channels were reliably present in cell-attached micropatches. This allowed resolution of both single channel events and averaged behavior. Both positive and negative pressures (up to 50mmHg) produced visible patch distention, an increase in patch current at all voltages and large hyperpolarizing shifts in steady-state voltage-sensitivity of activation and inactivation. From voltage dependence of activation at rest (V1/2 = −30mV at 0mmHg), pressure resulted in graded shifts of V1/2 for activation and inactivation of −0.71mV/mmHg and −0.72mV/mmHg, respectively. Channel kinetics were predictably affected by the voltage shifts, but channel opening and fast inactivation were otherwise unaffected by pressure. Single channel traces showed that unitary conductance was unaffected, rather peak currents appeared to increase due to an increase in the number of active channels in the patch. These effects were minimally reversible for as long as 30 minutes after a single stretch stimulus. Patch excision resulted in an immediate shift of activation V1/2=-75mV and loss of stretch sensitivity. Application of the inhibitor of actin polymerization, cytochalasin D, diminished sensitivity to stretch (−0.42mV/mmHg). Our work demonstrates that mechanical stress at physiologically relevant levels affects voltage sensing of Nav1.5 channels, without affecting the pore, channel gate and fast inactivation. Supported by NIH DK52766.