The N-terminal Domain Tethers the Voltage-gated Calcium Channel β2e- subunit to the Plasma Membrane via Electrostatic and Hydrophobic Interactions

Background: Membrane recruitment of the calcium channel (2e)-subunit remains elusive. Results: Charge neutralization of (2e) N-terminal residues abolishes its lipid-binding and surface-targeting abilities and its slow inactivation-conferring phenotype. Conclusion: The (2e)-subunit anchors to the membrane via electrostatic interactions likely involving an N-terminal in-plane -helix and a tryptophan side-chain penetration. Significance: A novel mechanism for -subunit membrane-anchoring supporting slow inactivation is presented. The -subunit associates with the (1) pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four -subunits are encoded by four different genes with multiple splice variants. Only two members of this family, (2a) and (2e), associate with the plasma membrane in the absence of the (1)-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of (2a) promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which (2e) anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in (2e) encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic (1b) isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type (2e), but not with a (2e) membrane association-deficient mutant, slows down inactivation. We propose that a predicted -helix within this domain orienting parallel to the membrane tethers the (2e)-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the -subunit family that also sustains slowed inactivation.