Molecular basis of the PIP2-dependent regulation of Cav2.2 channel and its modulation by Cav 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 beta subunit-dependent manner. However, the molecular mechanism by which the beta subunits control the PIP2 sensitivity of Ca-v channels remains unclear. By engineering various alpha 1B and beta 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-v 2.2 channels. Moreover, they were distinctly engaged in the regulation of channel gating depending on the coupled Ca-v beta 2 subunits. The membrane-anchored beta subunit abolished the PIP2 interaction of the phospholipid-binding site in the I-II loop, leading to lower PIP2 sensitivity of Ca-v 2.2 channels. By contrast, PIP2 interacted with the basic residues in the S4(II )domain of Ca-v 2.2 channels regardless of beta 2 isotype. Our data demonstrated that the anchoring properties of Ca-v beta 2 subunits to the plasma membrane determine the biophysical states of Ca-v 2.2 channels by regulating PIP, coupling to the nonspecific phospholipid-binding site in the I-II loop.