Mechanism of sodium channel NaV1.9 potentiation by G-protein signaling

Abstract

Tetrodotoxin (TTX)-resistant voltage-gated Na (NaV) channels have been implicated in nociception. In particular, NaV1.9 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 NaV1.9, we elucidated the biophysical mechanisms responsible for potentiation of channel activity by G-protein signaling to better understand the response to inflammatory mediators. Heterologous NaV1.9 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 (Po) 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 GTPγS had no effect on single-channel amplitude but caused an increased mean open time and greater Po compared with recordings made in the absence of GTPγS. We conclude that G-protein activation potentiates human NaV1.9 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 NaV1.9 potentiation by G-protein signaling during inflammation and provide a cellular platform useful for the discovery of NaV1.9 modulators with potential utility in treating inflammatory pain.

Heterologous expression of NaV1.9 chimeras in various cell systems

“…Transfected cells were detected via anti-CD8-coated microbeads (Dynabeads, Invitrogen, Karlsruhe, Germany) according to [15]. Cells expressing Na V 1.9 or Na V 1.9_C4 were kept at 28°C for a period of about 24 h after transfection according to [38].…”

“…Although feasible, this approach still leaves large Na V 1.8 currents as background signal. A valuable alternative was pointed out recently by Vanoye et al [38] who investigated the regulation of Na V 1.9 via the PKC pathway upon heterologous expression in the DRG hybridoma cell line ND7/23. But even in this case, endogenous TTX-sensitive Na V channels have to be blocked during current recording, and only very limited expression is achieved even when cells are cultivated at low temperature (28°C) after transfection.…”

Sodium channel NaV1.9 mutations associated with insensitivity to pain dampen neuronal excitability

“…The L811P and L1302F mutations were introduced into full-length human Na V 1.9 (GenBank accession number NP_0554858.2) cDNA including a C-terminal triple FLAG epitope as previously described (22). All recombinant cDNAs were sequenced in their entirety to confirm the presence of the intended modifications and the absence of unwanted mutations.…”

“…Stable Na V 1.9-expressing cells were selected with puromycin (3 g/ml; Gibco, Invitrogen, Thermo Fisher Scientific), and individual cell colonies were isolated by limiting dilution. The ND7/23 cell line stably expressing human WT Na V 1.9 was previously reported (22).…”

“…These physiological contributions of Na V 1.9 may stem from its unique biophysical properties. Specifically, Na V 1.9 exhibits voltage dependence of activation and inactivation that overlap near the resting membrane potential (RMP), slow inactivation gating kinetics, and a very large, persistent current (11,(20)(21)(22)(23)(24). These properties have been hypothesized to enable Na V 1.9 to regulate the threshold for excitability of peripheral nociceptive sensory neurons by modulating both the RMP and responses to subthreshold stimuli (14,15,17,23).…”