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  • Title: Dihydropyridine-sensitive calcium channels from skeletal muscle. I. Roles of subunits in channel activity.
    Author: Gutierrez LM, Brawley RM, Hosey MM.
    Journal: J Biol Chem; 1991 Sep 05; 266(25):16387-94. PubMed ID: 1715865.
    Abstract:
    Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are hetero-oligomeric proteins. Little is known about the functional roles of the various subunits, except that the alpha 1 subunit is the essential channel unit. We have reconstituted both partially purified holomeric channels and the separated subunits into liposomes and measured their properties using an assay based on the Ca2+ indicator dye fluo-3. The holomeric channels exhibited Ca2+ influx that was sensitive to membrane potential achieved by the addition of valinomycin in the presence of a K+ gradient. Dissipation of the K+ gradient resulted in the loss of the valinomycin-sensitive Ca2+ flux. In addition, the reconstituted channels were: 1) activated by the dihydropyridine Ca2+ channel activator Bay K 8644 in a dose-dependent manner with a Kd of 20 nM; 2) inhibited by various types of Ca2+ channel inhibitors including the dihydropyridine (+)-PN 200-110, the phenylalkylamine verapamil, and the benzothiazepine d-cis-diltiazem; and 3) modulated in a stereoselective manner by the enantiomers of the dihydropyridine S-202-791. The purified channels used in this work possessed an alpha 1 subunit of 165 kDa and did not appear to contain a larger alpha 1 subunit of approximately 210 kDa, suggesting that channel activity with properties similar to those observed in intact cells can be supported with an alpha 1 subunit of 165 kDa. Reconstituted channels that were 85% depleted in the alpha 2/delta subunits showed a significant decrease in the initial rate of Ca2+ influx induced by valinomycin, but retained responsiveness to Bay K 8644 and (+)-PN 200-110. When the separated alpha 2 and delta subunits were added back to the alpha 1 subunit-containing preparation, the channels exhibited their normal rate of Ca2+ influx. These results demonstrated that the dihydropyridine-sensitive Ca2+ channels from skeletal muscle require the presence of the alpha 2.gamma complex in stoichiometric amounts to exhibit full activity.
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