CALCIUM REGULATION OF SKELETAL-MUSCLE THIN FILAMENT MOTILITY IN-VITRO

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fas t skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labe ling was used to visualize the filaments by epifluorescence, and assay s were conducted at 30 degrees C and at ionic strengths near the physi ological range. Regulated thin filaments exhibited well-regulated beha vior when tropomyosin and troponin were added to the motility solution s because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+] , whereas the number of regulated thin filaments moving was more steep ly regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward high er [Ca2+] and was steepest at the highest ionic strength studied (0.14 M Gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no e ffect on the Ca2+ dependence of speed or number of filaments moving. T hese conclusions hold for five different methods used to analyze the d ata, indicating that the conclusions are robust. The force-pCa relatio nship (pCa = -log(10)[Ca2+]) for rabbit psoas skinned fibers taken und er similar conditions of temperature and solution composition (0.14 M Gamma/2) paralleled the speed-pCa relationship for the regulated filam ents in the in vitro motility assay. Comparison of motility results wi th the force-pCa relationship in fibers suggests that relatively few c ross-bridges are needed to make filaments move, but many have to be cy cling to make the regulated filament move at maximum speed.