LIMITED CARBODIIMIDE DERIVATIZATION MODIFIES SOME FUNCTIONAL-PROPERTIES OF THE SARCOPLASMIC-RETICULUM CA2+ RELEASE CHANNEL

Sarcoplasmic reticulum membrane derived from the terminal cisternae re gion reacts with the carboxyl reagent N,N'-dicyclohexylcarbodiimide. T he extension of this reaction is dependent on the reagent/protein rati o. By using a low ratio (10 muM reagent and 1 mg of protein/mL), we ca n selectively prevent the closure of the 450-kDa Ca2+ channel. Rapid f iltration experiments indicate no alteration in the activating mechani sm of Ca2+ release induced by Ca2+ or Sr2+ whereas the Ca2+ efflux inh ibition by Ca2+, Mg2+, or ruthenium red disappears after the chemical treatment. The activating/inhibitory effect of ryanodine on the Ca2+ c hannel does not appear to be perturbed by N,N'-dicyclohexylcarbodiimid e. The negligible incorporation of the C-14 radioactive reagent to the 450-kDa band (the Ca2+ channel subunit) indicates the possibility of protein cross-linking in addition to simple derivatization. The functi onal alterations produced by this reagent suggest the presence of crit ical acidic residue(s) in a hydrophobic environment which are involved in the low-affinity cationic binding site. They can be tentatively as sociated with hydrophobic domains of the channel subunits contributing to the lining of the pore for Ca2+ release. The data also indicate th at the channel activation by micromolar Ca2+ occurs in a different pro tein domain which is carbodiimide-insensitive under the experimental c onditions tested.