title: Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).
abstract: We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.
authors.affiliation: Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.
authors.forename: S R
authors.initials: SR
authors.lastname: Chen
authors.forename: P
authors.initials: P
authors.lastname: Leong
authors.forename: J P
authors.initials: JP
authors.lastname: Imredy
authors.forename: C
authors.initials: C
authors.lastname: Bartlett
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004553:Electric Conductivity; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers; D008274:Magnesium / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D008051:Lipid Bilayers; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium