Altered voltage-dependence revealed in an inactivation-deficient mutant of Nav1.4 via optogenetic PI(4,5)P2 dephosphorylation.

Abstract

Voltage-gated sodium (NaV) channels are a key membrane protein of most excitable cells that activate upon depolarization, which initiates an action potential by rapid influx of Na+ ions. Voltage-dependent activation is followed by fast inactivation, which confers the refractory period for excitable tissues. Because the gating processes of NaV channels are mechanically coupled, changes in the fast inactivation process also alter their voltage-dependent activation properties. Most ion channels are regulated by membrane phospholipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2), however the PI(4,5)P2 modulation of NaV channels has not been determined. The mutant channel NaV1.4-L435W-L437C-A438W (WCW) presents significantly attenuated fast-inactivation and expresses robustly in Xenopus oocytes. To study the effects of PI(4,5)P2 dephosphorylation on NaV1.4 activation in channels with attenuated fast inactivation, we expressed NaV1.4-WCW in Xenopus oocytes and recorded currents using two-electrode voltage-clamp (TEVC). To dephosphorylate PI(4,5)P2, we co-expressed 4’ or 5’ phospholipid phosphatases that are recruited to the cell membrane upon exposure to blue light (λ=470 nm). 4’ dephosphorylation of PI(4,5)P2 produced an 8 ± 2% increase in peak NaV1.4 WCW channel currents at a depolarizing pulse to −20 mV. Significantly greater channel currents at −15 mV and −25 mV were also observed. Similarly, 5’ dephosphorylation of PI(4,5)P2 resulted in a 15 ± 2% increase in Nav1.4 WCW peak channel currents. Significantly greater channel currents were observed in the voltage range from −20 mV to +20 mV. Although the peak currents were augmented, the half-maximal activation voltage (V1/2) and slope factor (Kact) of the WCW mutant were both unaffected by either dephosphorylation, suggesting that the altered voltage-dependence of this channel mutant is at least partially due to the effect of PI(4,5)P2 dephosphorylation on fast inactivation.