Purification of the putative calcium channel from skeletal muscle with the aid of [3H]-nimodipine binding
High affinity (KD=1.5 nmol/l) binding sites for the potent 1.4-dihydropyridine calcium channel blocker [3H]-nimodipine have been found in guinea-pig skeletal muscle homogenates. These sites could be enriched by differential centrifugation in a crude microsomal fraction. In the microsomal fraction the density of binding sites was 2pMol per mg of protein. The pharmacological profile of the [3H]-nimodipine binding sites suggests that they are part of the putative calcium channel and that [3H]-nimodipine binding can be used as a marker for these ionic pores.
In guinea-pig brain membranes [3H]-nimodipine labels a single class of sites with a KD of 0.6 nmol/l and d-cis-diltiazem decreases the KD by a factor of three without a change in the maximum number of receptors. In contrast d-cis-diltiazem (at the optimal concentration of 10 μmol/l) increases the density of the sites in guinea-pig skeletal muscle available for [3H]-nimodipine with high affinity and the KD decreases marginally to 1.0 nmol/l. These effects of d-cis-diltiazem are stereospecific since the pharmacologically inactive diastereoisomer l-cis-diltiazem does not stimulate [3H]-nimodipine binding, but is inhibitory, albeit at much higher concentrations. It is concluded that a significant fraction of the putative calcium channels has a KD of >50 nmol/l for [3H]-nimodipine, and that d-cis-diltiazem can increase the affinity of this subpopulation for [3H]-nimodipine so that they are detectable in ligand binding experiments.
The binding sites of [3H]-nimodipine have been purified from the crude microsomal pellet by means of sucrose gradient centrifugation. [3H]-nimodipine binding copurifies with (Na+, K+)-ATP' ase and [3H]-ouabain binding and is enriched in a vesicular fraction (by a factor of 30–60 fold) of low bouyant density [<25% (w/w) sucrose], with a ratio of (Na+, K+)-ATP'ase to Ca2+-ATP'ase activity of 0.77.
Biochemical and electron microscopic examination suggests that a specialized structure of the sarcolemma, possibly the transverse tubule, is the subcellular locus for the [3H]-nimodipine binding site. Since the density of the drug receptors in this purified preparation is extremely high and exceeds that reported for [3H]-saxitoxin binding sites (a specific sodium channel label) by a factor of 4–10 (with respect to most highly purified skeletal muscle membrane isolated), the isolation and purification of the putative calcium channel from skeletal muscle is feasable.
Our results confirm recent findings with biophysical methods, on the presence of calcium channels which are blocked by (±) D-600, d-cis-diltiazem and the 1,4-dihydropyridine nifedipine in skeletal muscle. The data are discussed in the context of the possible physiological role of calcium channels located in transverse tubules.
Key wordsSkeletal muscle Calcium channel blocker [3H]-Nimodipine Isolation
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