Modulation of Ca2+ exchange with the Ca(2+)-specific regulatory sites of troponin C.
Calcium (Ca2+) binding to the N-terminal Ca(2+)-specific sites on troponin C (TnC) regulate the contraction-relaxation cycle of skeletal muscle. A mutant TnC (F29W) and dansylaziridine-labeled TnC undergo large fluorescence increases when Ca2+ binds to their Ca(2+)-specific sites (half-maximal at pCa 5.8). Calmidazolium and the additional mutation of Met-82 to Gln (F29W,M82Q) increased Ca2+ affinity at these Ca2+ sites by approximately 4-fold (half-maximal at pCa approximately 6.4). Calmidazolium and the M82Q mutation decreased the rate of Ca2+ dissociation from the Ca(2+)-specific sites approximately 3.4-fold (from approximately 462 +/- 84/s to approximately 138 +/- 30/s) at 22 degrees C. Ca2+ associated with the Ca(2+)-specific sites of these proteins at 1-2 x 10(8) M-1 s-1 at 4 degrees C. These drug- and mutation-induced increases in Ca2+ affinity occur solely from large decreases in the Ca2+ off-rate without an effect on the Ca2+ on-rate. Thus, Ca2+ can bind to the Ca(2+)-specific sites of TnC as rapidly as it can diffuse to the protein, consistent with the extreme speed of skeletal muscle contraction. Drugs and/or site-directed mutagenesis can modify the Ca2+ sensitivity and the rate of Ca2+ exchange with TnC's Ca(2+)-specific sites to perhaps alter the rate of relaxation and/or the rate of rise of tension.
Johnson
J D
JD
Ohio State University Medical Center, Department of Medical Biochemistry, Columbus 43210.
Nakkula
R J
RJ
Vasulka
C
C
Smillie
L B
LB
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D014364:Tryptophan / Q000737:chemistry
D002135:Calcium-Binding Proteins; D007093:Imidazoles; D014336:Troponin; D019209:Troponin C; C031938:calmidazolium; D014364:Tryptophan; D002118:Calcium