Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) plays a crucial role in the gating of voltage-gated Ca 2+ (Ca V ) channels; however, the molecular mechanisms regulating its influence on these channels are not well understood. Recent studies have identified potential PIP 2 binding motifs in the S0 II and the voltage sensor S4 II of the N-type Ca V 2.2 channel. This study investigates the roles of these PIP 2 binding sites in modulating PIP 2 sensitivity and channel gating in the L-type Ca V 1.2 channel. To explore this, we neutralized four basic residues (R507, R508, R511, and R514) in S0 II , creating the AAAA mutant. This mutation resulted in decreased current density, a leftward shift in the activation curve, and loss of PIP 2 sensitivity. We further identified specific residues in S0 II involved in PIP 2 regulation. The R511A and R514A mutants retained minimal PIP 2 sensitivity; however, only R514A exhibited a leftward shift similar to the AAAA mutant. In contrast, the R508A and R507A mutants showed no significant effects, suggesting a specific interaction between residue R514 in the S0 II domain and PIP 2 . To assess the modulation of Ca V 1.2 by other phosphoinositide species, we conducted a chemically inducible dimerization assay. In this assay, rapamycin inhibited Ca V 1.2 currents more effectively than Dr-VSP in cells expressing pseudojanin, a polyphosphatase that converts PIP 2 to PI. The current inhibition observed with pseudojanin was comparable to the combined effects of Inpp5e (a 5’-phosphatase) and Sac1 (a 4’-phosphatase), indicating that PI4P also contributes to the regulation of Ca V 1.2. Additionally, phosphoinositide depletion consistently induced left-shifted activation curves. Finally, mutations of two basic residues on S4 II did not affect PIP 2 sensitivity. Overall, our findings suggest that Ca V 1.2 is predominantly regulated by PIP 2 at S0 II , which acts as an inhibitory site, with the putative PIP 2 binding region on S0 II potentially interacting electrostatically with both PIP 2 and PI4P.
Bae et al. (Sun,) studied this question.