Molecular basis of the PIP-dependent regulation of Ca2.2 channel and its modulation by Ca β subunits.

High-voltage-activated Ca (Ca) channels that adjust Ca influx upon membrane depolarization are differentially regulated by phosphatidylinositol 4,5-bisphosphate (PIP) in an auxiliary Ca β subunit-dependent manner. However, the molecular mechanism by which the β subunits control the PIP sensitivity of Ca channels remains unclear. By engineering various α1B and β constructs in tsA-201 cells, we reported that at least two PIP-binding sites, including the polybasic residues at the C-terminal end of I-II loop and the binding pocket in S4 domain, exist in the Ca2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled Ca β2 subunits. The membrane-anchored β subunit abolished the PIP interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP sensitivity of Ca2.2 channels. By contrast, PIP interacted with the basic residues in the S4 domain of Ca2.2 channels regardless of β2 isotype. Our data demonstrated that the anchoring properties of Ca β2 subunits to the plasma membrane determine the biophysical states of Ca2.2 channels by regulating PIP coupling to the nonspecific phospholipid-binding site in the I-II loop.