Electrostatic Association of Beta-Subunits to Membrane Reduces the PIP2 Sensitivity of Ca2+ Channels

Abstract

High voltage-gated Ca2+ (CaV) channels are protein complexes mostly containing α1, β and α2δ subunits. G protein-coupled receptors modulate these channels through diverse pathways. Recently, membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) was identified as a key regulator of these channels and sensitivity to PIP2 exclusively dependent on CaV β-subunits. using mouse β2a (mβ2a) subunit, we report that simple binding of this subunit to the membrane surface is sufficient for reducing PIP2 sensitivity. Compared to rat β2a (rb2a) subunits which are distributed on the plasma membrane following post-translational palmitoylation, mβ2a displayed the same distribution without any lipid modification, merely through its own N-terminus. Multi-alignment analysis between rβ2a and mβ2a revealed 98% sequence homology with main differences in N-terminus. To characterize the contribution of the N-terminus to mβ2a membrane targeting, the 23 amino acids of this region was sequentially deleted and membrane targeting and current properties of these mutants were measured. The results demonstrate the last basic amino acid, K2 residue to be critical for membrane distribution of β subunits and slow inactivation of Cav current. We also found W5 to be important for the membrane targeting of mβ2a subunit. Finally, binding of mβ2a subunit was diminished upon receptor stimulation, suggesting the involvement of electrostatic interaction during binding of mβ2a subunit to lipids. Consequently, we propose that the N-terminus of mβ2a, without lipidation, still plays a key role in membrane targeting and this interaction determines the low sensitivity of CaV channels to PIP2 depletion. Taken together, the results validate that lipidation of β subunit is not a critical factor for the reduction of CaV regulation by PIP2.