MODULATION OF CA2-SPECIFIC REGULATORY SITES OF TROPONIN-C( EXCHANGE WITH THE CA2+)

Calcium (Ca2+) binding to the N-terminal Ca2+-specific sites on tropon in C (TnC) regulate the contraction-relaxation cycle of skeletal muscl e. A mutant TnC (F29W) and dansylaziridine-labeled TnC undergo large f luorescence increases when Ca2+ binds to their Ca2+-specific sites (ha lf-maximal at pCa 5.8). calmidazolium and the additional mutation of M et-82 to Gln (F29W,M82Q) increased Ca2+ affinity at these Ca2+ sites b y similar to 4-fold (half-maximal at pCa similar to 6.4). Calmidazoliu m and the M82Q mutation decreased the rate of Ca2+ dissociation from t he Ca2+-specific sites similar to 3.4-fold (from similar to 462 +/- 84 /s to similar to 138 +/- 30/s) at 22 degrees C. Ca2+ associated with t he Ca2+-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+ affin ity occur solely from large decreases in the Ca2+ off-rate without an effect on the Ca2+ on rate. Thus, Ca2+ can bind to the Ca2+-specific s ites of TnC as rapidly as it can diffuse to the protein, consistent wi th 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 Ca2+-specific sites to perhaps alter the rate of relaxation and/or the rate of rise of tension.