CONFORMATIONAL-CHANGES IN THE JUNCTIONAL FOOT PROTEIN CA2+ RELEASE CHANNEL MEDIATE DEPOLARIZATION-INDUCED CA2+ RELEASE FROM SARCOPLASMIC-RETICULUM

In an attempt to monitor the kinetic events occurring in the junctional foot protein (JFP) during excitation-contraction coupling, the JFP moiety of isolated triads was covalently labeled in a site-directed manner with methylcoumarin acetate (MCA) using a recently developed technique (Kang, J. J., Tarcsafalvi, A., Carlos, A. D., Fujimoto, E., Shahrokh, Z., Thevenin, B. J. M., Shohet, S. B., and Ikemoto, N. (1992) Biochemistry 31, 3288-3293). Chemical depolarization of the transverse tubular system (T-tubule) moiety of labeled triads after appropriate priming induced first a rapid increase of the fluorescence intensity of the JFP-bound MCA probe, and then sarcoplasmic reticulum (SR) Ca2+ release. Upon increasing the magnitude of T-tubule depolarization by increasing the degree of ionic replacement, both the amplitude of the MCA fluorescence change and the amount of released Ca2+ increased in parallel. Blockers of T-tubuleto-SR communication, such as nimodipine and low concentration of neomycin, inhibited both the MCA fluorescence change and the SR Ca2+ release. In contrast, the release blocking concentration of Mg2+ (2 mM) inhibited only SR Ca2+ release without affecting the fluorescence change. These results suggest that upon T-tubule depolarization the original state of the JFP (R) isomerizes to an activated state with higher MCA fluorescence (*R), which in turn changes into a subsequent state in which the release channel is open (*R(0)):R-->R-->R(0).