Mechanism of sodium channel Na_V1.9 potentiation by G-protein signaling

Tetrodotoxin (TTX)-resistant voltage-gated Na (NaV) channels have been implicated in nociception. In particular, Na_V1.9 contributes to expression of persistent Na current in small diameter, nociceptive sensory neurons in dorsal root ganglia and is required for inflammatory pain sensation. Using ND7/23 cells stably expressing human Na_V1.9, we elucidated the biophysical mechanisms responsible for potentiation of channel activity by G-protein signaling to better understand the response to inflammatory mediators. Heterologous Na_V1.9 expression evoked TTX-resistant Na current with peak activation at 40 mV with extensive overlap in voltage dependence of activation and inactivation. Inactivation kinetics were slow and incomplete, giving rise to large persistent Na currents. Single-channel recording demonstrated long openings and correspondingly high open probability (P_o) accounting for the large persistent current amplitude. Channels exposed to intracellular GTPγS, a proxy for G-protein signaling, exhibited twofold greater current density, slowing of inactivation, and a depolarizing shift in voltage dependence of inactivation but no change in activation voltage dependence. At the single-channel level, intracellular GTγS had no effect on single-channel amplitude but caused an increased mean open time and greater P_o compared with recordings made in the absence of GTγS. We conclude that G-protein activation potentiates human Na_V1.9 activity by increasing channel open probability and mean open time, causing the larger peak and persistent current, respectively. Our results advance our understanding about the mechanism of Na_V1.9 potentiation by G-protein signaling during inflammation and provide a cellular platform useful for the discovery of Na_V1.9 modulators with potential utility in treating inflammatory pain.