Membrane-localized β-subunits alter the PIP2 regulation of high-voltage activated Ca2+ channels

The beta-subunits of voltage-gated Ca2+ (Ca-V) channels regulate the functional expression and several biophysical properties of high-voltage-activated Ca-V channels. We find that Ca-V beta-subunits also determine channel regulation by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). When Ca(V)1.3, -2.1, or -2.2 channels are cotransfected with the beta 3-subunit, a cytosolic protein, they can be inhibited by activating a voltage-sensitive lipid phosphatase to deplete PIP2. When these channels are coexpressed with a beta 2a-subunit, a palmitoylated peripheral membrane protein, the inhibition is much smaller. PIP2 sensitivity could be increased by disabling the two palmitoylation sites in the beta 2a-subunit. To further test effects of membrane targeting of Ca-V beta-subunits on PIP2 regulation, the N terminus of Lyn was ligated onto the cytosolic beta 3-subunit to confer lipidation. This chimera, like the Ca-V beta 2a-subunit, displayed plasma membrane localization, slowed the inactivation of Ca(V)2.2 channels, and increased the current density. In addition, the Lyn-beta 3 subunit significantly decreased Ca-V channel inhibition by PIP2 depletion. Evidently lipidation and membrane anchoring of Ca-V beta-subunits compete with the PIP2 regulation of high-voltage-activated Ca-V channels. Compared with expression with Ca-V beta 3-subunits alone, inhibition of Ca(V)2.2 channels by PIP2 depletion could be significantly attenuated when beta 2a was coexpressed with beta 3. Our data suggest that the Ca-V currents in neurons would be regulated by membrane PIP2 to a degree that depends on their endogenous beta-subunit combinations.