CA2-SITES OF THE RYANODINE RECEPTOR CA2+ RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM - LOW-AFFINITY BINDING SITE(S) AS PROBED BY TERBIUM FLUORESCENCE( BINDING)

Fluorescence spectroscopy has been used to study the interaction of Tb 3+ (as a Ca2+ analog) with the purified ryanodine receptor (RyR)/Ca2release channel of skeletal muscle sarcoplasmic reticulum. Tb3+ replac es Ca2+ in both the high- and the low-affinity sites. Occupation of th e low-affinity site (inhibitory), but not of the high-affinity Ca2+ bi nding site (activating), by Tb3+ results in a strong enhanced green fl uorescence (at 543 nm) and in an inhibition of ryanodine binding. The Tb3+ concentrations required for half-maximal enhanced fluorescence an d inhibition of ryanodine binding were: 22.5 +/- 2.5 mu M (n = 4) and 22.3 +/- 3.1 mu M (n = 2), respectively. Tb3+ appears to bind to the p rotein at two or more cooperative sites (n(H) = 2.4) and to dissociate from these sites with three different rate constants (K--1,K-1 = 361 +/- 250 min(-1) (n = 6); K--1,K-2 = 0.45 +/- 0.22 min(-1) (n = 11); K- -1,K-3 = 0.011 +/- 0.013 min(-1) (n = 7). The enhancement in Tb3+ fluo rescence is very fast (K-1 >> 5 x 10(5) M(-1).min(-1)), and it is quen ched by EGTA, La3+, or Ca2+ addition. About 20% of the bound Tb3+ was not displaced by EGTA or Ca2+; suggesting its ''occlusion'' in the RyR . This is also reflected in the partially irreversible inhibition of r yanodine binding by Tb3+. Reconstitution of sarcoplasmic reticulum ves icles into a planar bilayer lipid membrane showed that the Ca2+ releas e channel was activated by submicromolar and inhibited by micromolar c oncentrations of Tb3+ and La3+. The Tb3+-activated channel showed an e nhancement of the open dwell time of the channel. The results suggest that RyR/Ca2+ release channel undergoes conformational changes due to Tb3+ binding to the low-affinity Ca2+ binding site, and this binding r esults in the closing of the Ca2+ release channel.