Regulation of the RYR1 and RYR2 Ca2+ release channel isoforms by Ca2+-insensitive mutants of calmodulin.
Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.
Fruen
Bradley R
BR
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Black
D J
DJ
Bloomquist
Rachel A
RA
Bardy
Jennifer M
JM
Johnson
J David
JD
Louis
Charles F
CF
Balog
Edward M
EM
D000409:Alanine / Q000235:genetics; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002121:Calcium Channel Blockers / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine
D002121:Calcium Channel Blockers; D002147:Calmodulin; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D018698:Glutamic Acid; D000409:Alanine; D002118:Calcium