title: Cardiac ryanodine receptor: Selectivity for alkaline earth metal cations points to the EF-hand nature of luminal binding sites.
abstract: A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca(2+) is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M(2+): Mg(2+), Ca(2+), Sr(2+) or Ba(2+)). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M(2+). Moreover, we performed competition experiments by varying the concentration of luminal M(2+) (Mg(2+), Sr(2+) or Ba(2+)) from 8 mM to 53 mM and investigated its ability to compete with 1mM luminal Ca(2+). We demonstrate that all tested M(2+) bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca(2+)>Sr(2+)>Mg(2+)~Ba(2+), showing a strong correlation with the M(2+) affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function.
authors: Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Health Sciences Pavilion, 840 05, Bratislava, Slovak Republic. Electronic address: jana.gaburjakova@savba.sk.
authors: Jana
authors: J
authors: Gaburjakova
authors: Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Health Sciences Pavilion, 840 05, Bratislava, Slovak Republic. Electronic address: marta.gaburjakova@savba.sk.
authors: Marta
authors: M
authors: Gaburjakova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000927:Anticonvulsants / Q000378:metabolism / Q000494:pharmacology*; D001464:Barium / Q000378:metabolism; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D020832:EF Hand Motifs; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008673:Metals, Alkaline Earth / Q000378:metabolism*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D010634:Phenobarbital / Q000378:metabolism / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013324:Strontium / Q000378:metabolism
chemical_list: D000927:Anticonvulsants; D002413:Cations, Divalent; D000697:Central Nervous System Stimulants; D008673:Metals, Alkaline Earth; D019837:Ryanodine Receptor Calcium Release Channel; D001464:Barium; D002110:Caffeine; D008274:Magnesium; D002118:Calcium; D010634:Phenobarbital; D013324:Strontium
keywords: Alkaline earth metal divalent; Cardiac muscle; Luminal regulation; Planar lipid bilayer; Ryanodine receptor