Molecular basis of the PIP₂-dependent regulation of Ca_V2.2 channel and its modulation by Ca_V β subunits.

High-voltage-activated Ca²⁺ (Ca_V) channels that adjust Ca²⁺ influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP₂) in an auxiliary Ca_V β subunit-dependent manner. However, the molecular mechanism by which the β subunits control the PIP₂ sensitivity of Ca_V channels remains unclear. By engineering various α1B and β constructs in tsA-201 cells, we reported that at least two PIP₂-binding sites, including the polybasic residues at the C-terminal end of I-II loop and the binding pocket in S4_II domain, exist in the Ca_V2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled Ca_V β2 subunits. The membrane-anchored β subunit abolished the PIP₂ interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP₂ sensitivity of Ca_V2.2 channels. By contrast, PIP₂ interacted with the basic residues in the S4_II domain of Ca_V2.2 channels regardless of β2 isotype. Our data demonstrated that the anchoring properties of Ca_V β2 subunits to the plasma membrane determine the biophysical states of Ca_V2.2 channels by regulating PIP₂ coupling to the nonspecific phospholipid-binding site in the I-II loop.