Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay.
A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.
Du
G G
GG
Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
Imredy
J P
JP
MacLennan
D H
DH
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D014316:Tritium
D018076:DNA, Complementary; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012433:Ryanodine; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium