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.