Importance of the different beta subunits in the membrane expression of the alpha1A and alpha2 calcium channel subunits: studies using a depolarization-sensitive alpha1A antibody.
The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.
Brice
N L
NL
Department of Pharmacology, Royal Free Hospital School of Medicine, London, UK.
Berrow
N S
NS
Campbell
V
V
Page
K M
KM
Brickley
K
K
Tedder
I
I
Dolphin
A C
AC
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity; D001921:Brain / Q000502:physiology; D019556:COS Cells; D015220:Calcium Channels / Q000096:biosynthesis / Q000276:immunology / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology; D005727:Ganglia, Spinal / Q000502:physiology*; D046911:Macromolecular Substances; D008564:Membrane Potentials; D009474:Neurons / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection
D000906:Antibodies; D015220:Calcium Channels; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins