title: The N-terminal domain tethers the voltage-gated calcium channel β2e-subunit to the plasma membrane via electrostatic and hydrophobic interactions.
abstract: The β-subunit associates with the α1 pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β2a and β2e, associate with the plasma membrane in the absence of the α1-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β1b 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 β2e, but not with a β2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β2e-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 β-subunit family that also sustains slowed inactivation.
authors: Institute of Complex Systems 4, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52425 Jülich.
authors: Erick
authors: E
authors: Miranda-Laferte
authors: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors: David
authors: D
authors: Ewers
authors: Institute of Complex Systems 4, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52425 Jülich.
authors: Raul E
authors: RE
authors: Guzman
authors: Institute of Complex Systems 4, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52425 Jülich.
authors: Nadine
authors: N
authors: Jordan
authors: Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors: Silke
authors: S
authors: Schmidt
authors: Institute of Complex Systems 4, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52425 Jülich. Electronic address: pa.hidalgo@fz-juelich.de.
authors: Patricia
authors: P
authors: Hidalgo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry*; D004594:Electrophysiology; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D014364:Tryptophan / Q000737:chemistry
chemical_list: C491527:Cacnb2 protein, rat; D020746:Calcium Channels, L-Type; D008055:Lipids; D008081:Liposomes; D014364:Tryptophan
keywords: Calcium Channels; Electrophysiology; Phospholipid; Plasma Membrane; Tryptophan