In squid axons the Ca2+i regulatory site of the Na+/Ca2+ exchanger is drastically modified by sulfhydryl blocking agents. Evidences that intracellular Ca2+i regulatory and transport sites are different

Abstract

We have explored the effect of the sulfhydryl group blocker p-chloromercuryphenylsulfonic acid (PCMBS) on Ca2+ and Na+ interactions with the Na+/Ca2+ exchanger in squid giant nerve fibers. Steady-state Na+o-dependent Ca2+ efflux (forward) and Na+i-dependent Ca2+ influx (reverse) were measured in internally dialyzed, voltage clamped squid axons. External PCMBS (0.5 mM, for 25-35 min) has no effect on the activation of Ca2+ efflux by Na+o, and Ca2+o or on the activatory external monovalent cation site. In contrast, when applied internally it drastically reduces the affinity of the Na+/Ca2+ exchanger towards Ca2+i ions without affecting its maximal rate of transport; in the presence of MgATP the K0.5 for Ca2+i activation of forward Na+/Ca2+ exchange increases from 1.5 microM to 95 microM; likewise the apparent affinity of the Ca2+i stimulation of the reversal exchange decreases 100-fold. Interestingly, no effect of PCMBS was found on the interactions between Na+i and Ca2+i ions with the internal transport site(s) (inhibition of Na+2o and Ca2+o-dependent Ca2+ efflux by Na+i). On the other hand, Na+i ions do not modify the interactions of Ca2+i with that site. Two important characteristics of the Ca2+i regulatory site are uncover in this work: (i) sulfhydryl groups are important in maintaining the integrity of the Ca2+ binding domain of the Ca2+i regulatory site and (ii) Na+i and Ca2+i regulatory, or Na+i and Ca2+i transporting sites, are different entities.