Abstract
Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na(v) 1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na(v) α-subtypes in neurons, Na(v) 1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na(v) 1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na(v) 1.8 and the TTX-S Na(v) 1.2 using a domain exchange strategy. Wild-type and chimeric Na(v) channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique. MrVIB (1 µM) reduced Na(v) 1.2 current amplitude to 69 ± 12%, whereas Na(v) 1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na(v) 1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na(v) 1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na(v) 1.2. Taken together, these results suggest that domain II of Na(v) 1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.
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