Properties of the Domain-II Voltage Sensor Determining the Function of Nav1.8 (SCN10A) Channels

Abstract

NaV1.8 voltage-gated sodium channels are primarily expressed in dorsal root ganglia neurons and are implicated in pain perception. One of their remarkable features is their activation at relatively depolarized membrane voltages (Va = −5.3±1.5 mV compared to −40.2±2.2 mV for skeletal muscle NaV1.4). Searching for the underlying molecular determinants, we constructed domain chimeras between rat NaV1.4 and NaV1.8 channels and assayed them with the whole-cell patch-clamp method after expression in Neuro-2A cells. While we could not obtain any current response with 8444, i.e. a construct with domain-I from NaV1.8 and the remaining domains from NaV1.4, analysis of chimeras 4844 (Va = −12.8±2.2 mV), 4484 (−40.4±1.5 mV) and 4448 (−25.7±2.0 mV) suggests that the depolarized activation voltage mainly results from domain-II. The voltage sensor in domain-II of NaV1.8 harbors a double KK motif in the S3/S4 linker (KK726 and KK747), a feature unique for NaV1.8 channels. Mutating the KK motifs to the corresponding residues of NaV1.4 results in a left-shift in activation for KK747NV (Va= −15.6±1.3 mV). The reverse mutations in NaV1.4, QG634KK and NV655KK, have a qualitatively opposite effect. Furthermore, the KK motifs seem to interfere with the μO-conotoxin MrVIA, which is known to interact with the voltage-sensor of domain II via a sensor-trapping mechanism. Mutants KK747NV and KK726QG-KK747NV are blocked by 400 nM MrVIA by 91±1% and 86±8% respectively, compared to 64±4% for NaV1.8 wild-type channels. In addition, toxin dissociation at +40 mV is about 1.5 times faster for KK726QG-KK747NV than for wild-type channels. Thus, the KK motifs in the S3/S4 linker of NaV1.8 domain-II voltage-sensor take part in channel gating compatible with the “paddle model” and provide a molecular explanation for the gating mechanism unique to NaV1.8 channels.