PIP₂ stabilizes Na_V1.5 gating and links receptor signaling to cardiac late sodium current

The cardiac sodium channel Na_V1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of Na_V1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (I_Na,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP₂) is a critical membrane cofactor that stabilizes Na_V1.5 gating. Acute PIP₂ depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased I_Na,L. These effects were prevented by augmenting intracellular PIP₂, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca² or PKC signaling. Unlike the skeletal-muscle isoform Na_V1.4, Na_V1.5 displayed PIP₂-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP₂-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP₂-contact network, produced elevated basal I_Na,L, showed enhanced sensitivity to PIP₂ depletion, and caused an approximately 30-fold reduction in apparent functional PIP₂ sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes Na_V1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP₂ axis to promote proarrhythmic late sodium current.