Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel

Abstract

Many excitable cells contain at least two different voltage-dependent Ca channels (L- and T-type)^1–3. The cardiac, slow, L-type Ca channel is further modulated by cyclic AMP-dependent phosphorylation^4–7, which increases the probability of it being open^5,7, and is readily blocked by Ca channel blockers including dihydro-pyridines and phenylalkylamines^8–11. The tritiated congeners of these blockers bind in vitro to sites which have the same pharmacological characteristics as those observed in vivo^l2–l4 that is, stereospecific and allosteric interaction between distinct sites. The dihydropyridine-binding site purified from skeletal muscle t-tubules¹⁵ contains three peptides of relative molecular mass (M_r) 142,000 (142K), 56K and 31K. The cAMP kinase incorporates one mol phosphate per mol of the 142K peptide and binding of (+)PN-200/110, a potent Ca antagonist, is allosterically affected by D-cis-diltiazem and verapamil. The purified dihydropyridine-receptor complex has also been incorporated into phospholipid bilayer membranes. Here, we show for the first time that the complex can be reconstituted to form a functional 20-pS Ca channel that retains the principal regulatory, biochemical and pharmacological properties of membrane-bound L-type Ca channels.