title: Single-channel characterization of the rabbit recombinant RyR2 reveals a novel inactivation property of physiological concentrations of ATP.
abstract: Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems.
authors.affiliation: Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Stewart
authors.forename: Lele
authors.initials: L
authors.lastname: Song
authors.forename: Simon M
authors.initials: SM
authors.lastname: Carter
authors.forename: Charalambos
authors.initials: C
authors.lastname: Sigalas
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Zaccai
authors.forename: Venkateswarlu
authors.initials: V
authors.lastname: Kanamarlapudi
authors.forename: Manjunatha B
authors.initials: MB
authors.lastname: Bhat
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeshima
authors.forename: Rebecca
authors.initials: R
authors.lastname: Sitsapesan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008024:Ligands; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology
chemical_list: D002121:Calcium Channel Blockers; D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D013498:Suramin; D000255:Adenosine Triphosphate; D002118:Calcium