Critical regulation of the voltage-gated sodium channel, Nav1.4, by PIP2.

Abstract

Voltage-gated sodium (NaV) channels are vital to the electrical signalling of cells. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting a timer on the excitability of tissues. NaV channels are protein complexes that consist of a large, pore forming alpha subunit and are often associated with auxiliary beta subunits. The alpha subunit is made up of four tandem repeat domains (DI-IV) that consist of six transmembrane segments (S1-S6) with S4 in each domain serving as its putative voltage sensor. The gating cycle of NaV channels is subject to tight regulation, with dysfunction leading to several disease states. Most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]; however, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Of the nine (NaV1.1 - NaV1.9) family members that have been identified and classified based on tissue distribution, we studied NaV1.4 which is the isoform primarily expressed in skeletal muscle. We used the optogenetically activated inositide phosphatase, pseudojanin, an enzymatic chimera of inositol polyphosphate 5 phosphatase E (INPP5E), which converts PI(4,5)P2 to PI4P and the S. cerevisiae sac1 phosphatase, which dephosphorylates PI(4)P, to evaluate the role of PI(4,5)P2 in NaV1.4 channel gating and regulation. Through a combination of patch-clamp recording and optogenetics we show that dephosphorylating PI(4,5)P2 shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials. PI(4,5)P2 depletion also augments the late current that persists after fast inactivation, a phenotype that is associated with several mutations that lead to myotonia. Through this work, we provide evidence that PI(4,5)P2 is a necessary co-factor in the gating of NaV1.4 channels.