Unique modulation of L-type Ca2+ channels by short auxiliary β1d subunit present in cardiac muscle

Recent studies have identified a growing diversity of splice variants of auxiliary Ca2+ channel Ca-v beta subunits. The Ca-v beta 1d isoform encodes a putative protein composed of the amino-terminal half of the full-length Ca-v beta(1) isoform and thus lacks the known high-affinity binding site that recognizes the Ca2+ channel alpha(1)-subunit, the alpha-binding pocket. The present study investigated whether the Ca-v beta(1d) subunit is expressed at the protein level in heart, and whether it exhibits any of the functional properties typical of full-length Ca-v beta subunits. On Western blots, an antibody directed against the unique carboxyl terminus of Ca-v beta(1d) identified a protein of the predicted molecular mass of 23 kDa from canine and human hearts. Immunocytochemistry and surface-membrane biotinylation experiments in transfected HEK-293 cells revealed that the full-length Ca-v beta(1b) subunit promoted membrane trafficking of the pore-forming alpha(1C) (Ca(v)1.2)subunit to the surface membrane, whereas the Ca-v beta(1d) subunit did not. Whole cell patch-clamp analysis of transfected HEK-293 cells demonstrated no effect of coexpression of the Ca-v beta(1d) with the alpha(1C)-subunit compared with the 15-fold larger currents and leftward shift in voltage-dependent activation induced by full-length Ca-v beta(1b) coexpression. In contrast, cell-attached patch single-channel studies demonstrated that coexpression of either Ca-v beta(1b) or Ca-v beta(1d) significantly increased mean open probability four- to fivefold relative to the alpha(1C)-channels alone, but only Ca-v beta(1b) coexpression increased the number of channels observed per patch. In conclusion, the Ca-v beta(1d) isoform is expressed in heart and can modulate the gating of L-type Ca2+ channels, but it does not promote membrane trafficking of the channel complex.