(−)‐[3H]Desmethoxyverapamil and (+)‐[3P]PN 200‐110 were employed to characterize phenylalkylamineselective and 1,4‐dihydropyridine‐selective receptors on purified Ca2+ channels from guinea‐pig skeletal muscle t‐tubules. In contrast to the membrane‐bound Ca2+ channel, d‐cis‐diltiazem (EC50 = 4.5 ± 1.7 μM) markedly stimulated the binding of (+)‐[3H]PN 200‐110 to the purified ionic pore. In the presence of 100 μM d‐cis‐diltiazem (which binds to the benzothiazepine‐selective receptors) the B max for (+)‐[3H]PN 200‐110 increased from 497 ± 81 to 1557 ± 43 pmol per mg protein, whereas the K d decreased from 8.8 ± 1.7 to 4.7 ± 1.8 nM at 25°C. P‐cis‐Diltiazem was inactive. (−)‐Desmethoxyverapamil, which is a negative heterotropic allosteric inhibitor of (+)‐[3H]IN 200‐110 binding to membrane‐bound channels, stimulated 1,4‐dihydropyridine binding to the isolated channel. (−)‐[3H]Desmethoxyverapamil binding was stimulated by antagonistic 1,4‐dihydropyridines [(+)‐PN 200‐110 ⪢(−)(CR)‐202‐791 ⪢(+)(4R)‐Bay K 8644] whereas the agonistic enantiomers (+)(S)‐202‐791 and (−)(4S)‐Bay K 8644 were inhibitory and (−)‐PN 200‐110 was inactive. The results indicate that three distinct drug‐receptor sites exist on the purified Ca2+ channel, two of which are shown by direct labelling to be reciprocally allosterically coupled.

The tritiated arylazido phenylalkylamine (-)-5- [(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5-trimethoxyphenyl)-2-isopropylvaleronitrile was synthesized and used to photoaffinity label the phenylalkylamine receptor of the membrane-bound and purified calcium channel from guinea-pig skeletal muscle transverse-tubule m~branes.The photoaffinity ligand binds reversibly to partially purified membranes with a K* of 2.0 Ir 0.5 nM and a B,, of 17.0 + 0.9 pmollmg protein. Binding is stereos~ifically regulated by all three classes of organic calcium channel drugs. A 155 kDa band was specifically photolabelled in transverse-tubule particulate and purified calcium channel preparations after ultraviolet irradiation. Additional minor labelled polypeptides (92,60 and 33 kDa) were only observed in membranes. The heterogeneous 155 kDa region of the purified channel was resolved into two distinct silver-stained polypeptides after reduction (i.e. 155 and 135 kDa). Only the 155 kDa polypeptide carries the photoaffinity label and it is concluded that the 135 kDa polypeptide (which migrates as a 165 kDa band under alkylating conditions) is not a highaffinity drug receptor carrying subunit of the skeletal muscle transver~-tubule L-type calcium channel.

The voltage-dependent calcium channel from guinea-pig skeletal muscle T-tubules has been isolated with a rapid, two-step purification procedure. Reversible postlabelling of the channel-linked 1,4-dihydropyridine receptor and stereoselective photolabelling as a novel approach were employed to assess purity. A 135-fold purification to a specific activity of 1311 f 194 pmol/mg protein (determined by reversible equilibrium binding with (+)-[3H]PN200-110) was achieved. Three polypeptides of 155 kDa, 65 kDa and 32 kDa were identified in the purified preparation. The 155-kDa band is a glycoprotein. The arylazide photoaffinity probe (-)-[3H]azidopine bound with high affinity to solubilized membranes (Kd = 0.7 f 0.2 nM) and highly purified fractions (Kd = 3

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