Molecular Basis of the Membrane Interaction of the [Beta]2e Subunit of Voltage-Gated Ca^sup 2+^ Channels

The auxiliary ... subunit plays an important role in the regulation of voltage-gated calcium (Ca...) channels. Recently, it was revealed that ...2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of ...-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the ...2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP...). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the ...2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated ...2e subunit on inactivation kinetics and regulation of Ca... channels by PIP... In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of ...2e (K2A/W5A) increased the PIP... sensitivity of Ca...2.2 and Ca...1.3 channels by ~3-fold compared with wild-type ...2e subunit. Together, our results suggest that membrane targeting of the ...2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-...2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels. (ProQuest: ... denotes formulae/symbols omitted.)