High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels

D. P. Rardon, D. C. Cefali, R. D. Mitchell, S. M. Seiler, L. R. Jones

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

Abstract

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent M(r) = 400,000-350,000, and bound [3H]ryanodine with a K(d) of 4.6 nM and a B(max) of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 ± 13 pS and a E(rev) of 42 ± 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 ± 1 pS [1.0 μM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 μM to 1 nM reduced the large conductance channel open time from 7 ± 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 ± 1% to 52 ± 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 μM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.

Original languageEnglish (US)
Pages (from-to)779-789
Number of pages11
JournalCirculation research
Volume64
Issue number4
DOIs
StatePublished - 1989

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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