Limited carbodiimide derivatization modifies some functional properties of the sarcoplasmic reticulum Ca2+ release channel.
Sarcoplasmic reticulum membrane derived from the terminal cisternae region reacts with the carboxyl reagent N,N'-dicyclohexylcarbodiimide. The extension of this reaction is dependent on the reagent/protein ratio. By using a low ratio (10 microM reagent and 1 mg of protein/mL), we can selectively prevent the closure of the 450-kDa Ca2+ channel. Rapid filtration experiments indicate no alteration in the activating mechanism of Ca2+ release induced by Ca2+ or Sr2+ whereas the Ca2+ efflux inhibition by Ca2+, Mg2+, or ruthenium red disappears after the chemical treatment. The activating/inhibitory effect of ryanodine on the Ca2+ channel does not appear to be perturbed by N,N'-dicyclohexylcarbodiimide. The negligible incorporation of the 14C radioactive reagent to the 450-kDa band (the Ca2+ channel subunit) indicates the possibility of protein cross-linking in addition to simple derivatization. The functional alterations produced by this reagent suggest the presence of critical acidic residue(s) in a hydrophobic environment which are involved in the low-affinity cationic binding site. They can be tentatively associated with hydrophobic domains of the channel subunits contributing to the lining of the pore for Ca2+ release. The data also indicate that the channel activation by micromolar Ca2+ occurs in a different protein domain which is carbodiimide-insensitive under the experimental conditions tested.
Martinez-Azorin
F
F
Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria, Universidad de Murcia, Spain.
Gomez-Fernandez
J C
JC
Fernandez-Belda
F
F
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D012428:Ruthenium / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013324:Strontium / Q000378:metabolism
D015220:Calcium Channels; D008565:Membrane Proteins; D012433:Ryanodine; D004024:Dicyclohexylcarbodiimide; D012428:Ruthenium; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium; D013324:Strontium