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

Calcium (Ca2+) binding to the N-terminal Ca2+-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 Ca2+-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 similar to 4-fold (half-maximal at pCa similar to 6.4). Calmidazolium and the M82Q mutation decreased the rate of Ca2+ dissociation from the 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 the 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+ 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 Ca2+-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 Ca2+-specific sites to perhaps alter the rate of relaxation and/or the rate of rise of tension.