Programme Contents

Identification of Nav1.8 channel domains responsible for μO-conotoxin MrVIB binding and channel biophysical properties

Voltage-gated sodium channels (VGSCs) are expressed primarily in excitable cells, such as central and peripheral neurons and muscle, and play a pivotal role in the initiation and propagation of action potentials. To date, nine subtypes of the pore-forming α subunit have been identified, each with a distinct tissue distribution, biophysical property and sensitivity to the neurotoxin tetrodotoxin (TTX). The 260 kDa α-subunits exhibit intracellular N- and C-termini and consist of four domains, each containing six membrane-spanning segments. Na_v1.8, a TTX-resistant subtype, is predominantly expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In contrast to TTX-sensitive α-subtypes, Na_v1.8 exhibits slower activation and inactivation kinetics and is inhibited by μO-conotoxin MrVIB from Conus marmoreus (Ekberg et al., 2006). To determine which domain confers Na_v1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels between Na_v1.8 and a TTX-sensitive Na_v1.2 that is expressed in the central nervous sytem. Wild type and chimeric channels were expressed in Xenopus oocytes and depolarization-induced Na⁺ currents were recorded using the two-electrode voltage clamp technique. Slow inactivation kinetics of Na_v1.8 was changed to fast kinetics when domain 1 and 2 was replaced by the corresponding domains of Na_v1.2; slow activation kinetics remained unaltered. MrVIB (1 μM) inhibits Na_v1.8 currents by 80% whereas no significant effect was observed on Na_v1.2 currents. A similar sensitivity to MrVIB was observed for Na_v1.2 /1.8 chimeras containing Na_v1.8 domain 2. In contrast, Na_v1.2 /1.8 chimeras containing Na_v1.2 domain 2 were insensitive to MrVIB. Taken together, these results suggest that domain 2 of Nav1.8 is critical for MrVIB binding and activity. A previous study on Na_v1.4 reported that MrVIB hinders the voltage sensor in domain 2 from activating and, hence, the channel from opening (Leipold et al., 2007). The binding of TTX may rely on a comparative mechanism since the presence of one or two binding sites within VGSCs Na_v1.8/1.2 chimeras had no effect compared to the wild type VGSC subtype Na_v1.8. All results were confirmed by a set of at least 10 cells/experiments.

Ekberg J, Jayamanne A, Vaughan CW, Aslan S, Thomas L, Mould J, Drinkwater R, Baker MD, Abrahamsen B, Wood JN, Adams DJ, Christie MJ, Lewis RJ. (2006) Proceedings of the National Academy of Sciences USA 103: 17030-5.

Leipold E, DeBie H, Zorn S, Borges A, Olivera BM, Terlau H, Heinemann SH. (2007) Channels (Austin) 1(4): 253-262.