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 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-tubule-to-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 (*Ro):R-->*R-->*Ro.
Boston Biomedical Research Institute, Massachusetts 02114.
M
M
Yano
R
R
el-Hayek
N
N
Ikemoto
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry* / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D008861:Microsomes / Q000378:metabolism; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D009553:Nimodipine / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D009553:Nimodipine; D009355:Neomycin; D008274:Magnesium; D002118:Calcium