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). The cardiac, slow, L-type Ca channel is further modulated by cyclic AMP-dependent phosphorylation, which increases the probability of it being open, and is readily blocked by Ca channel blockers including dihydropyridines and phenylalkylamines. The tritiated congeners of these blockers bind in vitro to sites which have the same pharmacological characteristics as those observed in vivo, that is, stereospecific and allosteric interaction between distinct sites. The dihydropyridine-binding site purified from skeletal muscle t-tubules contains three peptides of relative molecular mass (Mr) 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.

Tissue-specific expression of calcium channels

“…The purified complex contains four proteins (Figure 2) (Flockerzi et al 1986, Hymel et al 1988, Nunoki et al 1989, Mundiiia-Weilenmann et al 1991. The primary sequences of these proteins have been deduced by cloning their corresponding cDNAs from rabbit skeletal muscle (Tanabe et al 1987, Ellis et al 1988, Ruth et al 1989, Jay et al 1990.…”

A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel α1C subunit

“…It is widely believed that this modulation is due to phosphorylation of one of the subunits of the channel, catalyzed by PKA [2], and there is good, though not conclusive, evidence for such a direct mechanism in the SkM Ca 2+ channel. Of the four subunits of this channel (~IS, ~2[~, ~1 and 7), two (0~lS and ]31) are substrates for PKA-catalyzed phosphorylation [4][5][6], and the channel activity is enhanced by PKA in native cells and after reconstitution of purified channel subunits in artificial membranes [7][8][9].…”

Purification of a functional receptor for calcium‐channel blockers from rabbit skeletal‐muscle microsomes

“…It was therefore important to demonstrate that this purified receptor also retains the allosteric binding characteristics of the membrane-bound sites of calcium channel blockers. The data presented in this communication together with that of [24] suggest that the allosteric properties of the calcium-channel-blocker binding sites are an intrinsic property of the voltage-dependent 1-type calcium channel. and TSK DEAE-5PW columns were from Pharmacia and LKB, respectively.…”

“…The preparation used in this work has been successfully reconstituted as a voltage-regulated I-type calcium channel [24]. It is therefore quite conceivable that the allosteric nature of the binding of organic calcium channel blockers is an intrinsic property of this channel.…”

“…So far, neither the functionality nor the allosteric properties of the purified dihydropyridine receptors have been demonstrated. Recently, we have shown that the dihydropyridine receptor purified from rabbit skeletal muscle microsomes is a calcium channel which shows the basic electrophysiological and pharmacological properties of an 1-type channel [24]. It was therefore important to demonstrate that this purified receptor also retains the allosteric binding characteristics of the membrane-bound sites of calcium channel blockers.…”