Calmodulin interaction with the skeletal muscle sarcoplasmic reticulum calcium channel protein.
Studies were initiated to define the equilibria of calmodulin binding to the skeletal muscle sarcoplasmic reticulum (SR) Ca(2+)-release channel protein in native SR vesicles. Calmodulin affinity-labeling experiments indicated that the major calmodulin receptor in heavy SR preparations was a protein of M(r) > 450,000, corresponding to the Ca(2+)-release channel protein. [3H]Ryanodine-binding assays indicated 10.6 +/- 0.9 pmol of high-affinity ryanodine binding per milligram of SR protein. Wheat germ calmodulin was derivatized with rhodamine-x-maleimide. The affinity and binding capacity of the channel protein in SR vesicles for the derivatized calmodulin (Rh-CaM) were determined by fluorescence anisotropy in the presence of (1) 1 mM EGTA, (2) 0.1 mM CaCl2, and (3) 0.1 mM CaCl2 plus 1 mM MgCl2. In the presence of EGTA, Rh-CaM bound to the channel protein with a Kd of 8.6 +/- 0.8 nM and a Bmax of 229 +/- 7 pmol/mg, suggesting that calmodulin binds to the channel protein at [Ca2+] comparable to that in resting muscle. In the presence of 0.1 mM CaCl2, the binding equilibrium shifted to a two-site ligand-binding model; the high-affinity class of sites had a Bmax1 of 54 +/- 7 pmol/mg and a Kd1 of 4.3 +/- 1.1 nM, while the lower affinity class of sites had a Bmax2 of 166 +/- 28 pmol/mg and a Kd2 of 239 +/- 102 nM. In the presence of both Ca2+ and Mg2+, there was a further change in the Rh-CaM/channel protein interaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Yang
H C
HC
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824-1224.
Reedy
M M
MM
Burke
C L
CL
Strasburg
G M
GM
D000345:Affinity Labels; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005454:Fluorescence Polarization; D015636:Magnesium Chloride / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism*; D009994:Osmolar Concentration; D011817:Rabbits; D012235:Rhodamines; D012433:Ryanodine / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D015220:Calcium Channels; D002147:Calmodulin; D012235:Rhodamines; D015636:Magnesium Chloride; D012433:Ryanodine; D004533:Egtazic Acid; D002122:Calcium Chloride