Determinants for Membrane Targeting of Non-Palmitoylated Voltage-Gated Calcium Channel β-Subunit

The β-subunit of high-voltage activated calcium channels alters several aspects of channel function depending on its interaction partner, oligomeric state and intracellular location. Four non allelic β-subunit isoforms (β1 to β4), each including multiple splice variants, have been identified. For β2, five splice variants (β2a-β2e) have been characterized that differ only in the sequence and length (ranging from 16 to 70 amino acid residues) of their N-terminal regions. Despite the high degree of homology between these β2 splice variants, they exhibit different intracellular localization even in the absence of the α1 pore-forming subunit. While only β2a and β2e anchors to the plasma membrane, all the other β-isoforms (β1 to β3) and β2-variants variants are entirely cytoplasmic. Palmitoylation of two continuous N-terminal cysteine residues in β2a is responsible for plasma membrane targeting of this variant. However the determinants of membrane association in β2e remain to be elucidated. using a combination of confocal fluorescent microscopy, spectrofluorometric measurements and protein-lipids interaction assay we identify a cluster of positively charged residues in β2e that are required for lipid association and membrane targeting. Moreover, binding to negatively charged liposomes, but not to neutral ones, depends on the ionic strength. Mutation of the single N-terminal cysteine residue in β2e preserves its membrane localization. Our data suggests that electrostatic interactions participate in anchoring the β2e-subunit to the plasma membrane.