Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules

Biochemistry. 1986 Jun 3;25(11):3077-83. doi: 10.1021/bi00359a002.

Abstract

The purified calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubule membrane consists of three subunits: alpha with Mr 135 000, beta with Mr 50 000, and gamma with Mr 33 000. Purified receptor preparations were incorporated into phosphatidylcholine (PC) vesicles by addition of PC in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and removal of detergent by molecular sieve chromatography. Forty-five percent of the alpha, beta, and gamma polypeptides and the [3H]dihydropyridine/receptor complex were recovered in association with PC vesicles. The rate of dissociation of the purified and reconstituted dihydropyridine/receptor complex was identical with that in T-tubule membranes, and allosteric modulation by verapamil and diltiazem was retained. The reconstituted calcium antagonist receptor, when occupied by the calcium channel activator BAY K 8644, mediated specific 45Ca2+ and 133Ba2+ transport into the reconstituted vesicles. 45Ca2+ influx was blocked by the organic calcium antagonists PN200-110 (K0.5 = 0.2 microM), D600 (K0.5 = 1.0 microM), and verapamil (K0.5 = 1.5 microM) and by inorganic calcium channel antagonists (La3+ greater than Cd2+ greater than Ni2+ greater than Mg2+) as in intact T-tubules. A close quantitative correlation was observed between the presence of the alpha, beta, and gamma subunits of the calcium antagonist receptor and the ability to mediate 45Ca2+ or 133Ba2+ flux into reconstituted vesicles. Comparison of the number of reconstituted calcium antagonist receptors and functional channels supports the conclusion that only a few percent of the purified calcium antagonist receptor polypeptides are capable of mediating calcium transport as previously demonstrated for calcium antagonist receptors in intact T-tubules.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Barium / metabolism
  • Calcium / metabolism*
  • Calcium Channel Blockers / metabolism
  • Calcium Channel Blockers / pharmacology
  • Ion Channels / drug effects
  • Ion Channels / metabolism*
  • Isradipine
  • Kinetics
  • Liposomes
  • Macromolecular Substances
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism*
  • Microtubules / metabolism*
  • Molecular Weight
  • Muscles / metabolism*
  • Oxadiazoles / metabolism
  • Phosphatidylcholines

Substances

  • Calcium Channel Blockers
  • Ion Channels
  • Liposomes
  • Macromolecular Substances
  • Membrane Proteins
  • Oxadiazoles
  • Phosphatidylcholines
  • Barium
  • Calcium
  • Isradipine