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Voltage-gated sodium (NaV) channels are densely expressed in most excitable cells and activate in response to depolarization, causing a rapid influx of Na+ ions that initiates the action potential. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting the refractory period of excitable tissues. The gating cycle of NaV channels is subject to tight regulation, with perturbations leading to a range of pathophysiological states. The gating properties of 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. Here, we utilize optogenetics to activate specific, membrane-associated phosphoinositide (PI)-phosphatases that dephosphorylate PI(4,5)P2 while simultaneously recording NaV1.4 channel currents. We show that dephosphorylating PI(4,5)P2 left-shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials, augments the late current that persists after fast inactivation, and speeds the rate at which channels recover from fast inactivation. These effects are opposed by exogenous diC8PI(4,5)P2. We provide evidence that PI(4,5)P2 is a negative regulator that tunes the gating behavior of NaV1.4 channels.

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P 2 ). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP 2 , an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

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