Ca2+ binding sites of the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum. Low affinity binding site(s) as probed by terbium fluorescence.
Fluorescence spectroscopy has been used to study the interaction of Tb3+ (as a Ca2+ analog) with the purified ryanodine receptor (RyR)/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum. Tb3+ replaces Ca2+ in both the high- and the low-affinity sites. Occupation of the low-affinity site (inhibitory), but not of the high-affinity Ca2+ binding site (activating), by Tb3+ results in a strong enhanced green fluorescence (at 543 nm) and in an inhibition of ryanodine binding. The Tb3+ concentrations required for half-maximal enhanced fluorescence and inhibition of ryanodine binding were: 22.5 +/- 2.5 microM (n = 4) and 22.3 +/- 3.1 microM (n = 2), respectively. Tb3+ appears to bind to the protein at two or more cooperative sites (nH = 2.4) and to dissociate from these sites with three different rate constants (K-1,1 = 361 +/- 250 min-1 (n = 6); K-1,2 = 0.45 +/- 0.22 min-1 (n = 11); K-1,3 = 0.011 +/- 0.013 min-1 (n = 7). The enhancement in Tb3+ fluorescence is very fast (K1 >> 5 x 10(5) M-1.min-1), and it is quenched 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 ryanodine binding by Tb3+. Reconstitution of sarcoplasmic reticulum vesicles into a planar bilayer lipid membrane showed that the Ca2+ release channel was activated by submicromolar and inhibited by micromolar concentrations of Tb3+ and La3+. The Tb(3+)-activated channel showed an enhancement 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 results in the closing of the Ca2+ release channel.
Hadad
N
N
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Zable
A C
AC
Abramson
J J
JJ
Shoshan-Barmatz
V
V
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D005453:Fluorescence; D009124:Muscle Proteins / Q000378:metabolism*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013725:Terbium / Q000378:metabolism*
D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D013725:Terbium; D002118:Calcium