Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated L-type Ca2+ channel

Highly purified L-type Ca2+ channel complexes containing all five subunits (alpha (1), alpha (2), beta, gamma, and delta) and complexes of alpha (1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy, Projection images of negatively stained Ca2+ channels and alpha (1)-beta complexes were aligned, classified and averaged. The alpha (1)-beta Complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca2+ channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca2+ channel using a monoclonal antibody against the beta subunit showed that the alpha (1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca2+ channel exhibits an asymmetric arrangement of auxiliary subunits. (C) 2001 Academic Press.