A CONFORMATIONAL CHANGE IN THE JUNCTIONAL FOOT PROTEIN IS INVOLVED IN THE REGULATION OF CA2+ RELEASE FROM SARCOPLASMIC-RETICULUM - STUDIES ON POLYLYSINE-INDUCED CA2+ RELEASE

We investigated both conformational changes in the junctional foot protein (JFP) and Ca2+ release from sarcoplasmic reticulum (SR) in parallel after stimulation of triadic vesicles by the JFP-specific ligand, polylysine, To monitor protein conformational change, the JFP was labeled in a site-directed fashion with the fluorescent conformational probe methylcoumarin acetate (MCA) (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), The induction of SR Ca2+ release by polylysine produced a rapid increase in the fluorescence intensity of the JFP-bound MCA. The polylysine concentration dependence of the fluorescence change was essentially the same as that of Ca2+ release, suggesting that the two events are tightly coupled, However, the rate constant of MCA fluorescence change was much larger than that of Ca2+ release; i.e. the conformational change preceded Ca2+ release, Prevention of protein conformational change by lysine (0.2 M) inhibited Ca2+ release from SR, Inhibition of Ca2+ release by Mg2+ (5 mM), however, had little effect on the conformational change, These results suggest that binding of polylysine to the JFP produces conformational changes in the protein, which in turn activates the Ca2+ channel, leading to Ca2+ release from the SR.