IN SQUID AXONS THE CA(I)2+ REGULATORY SITE OF THE NA+/CA2+ EXCHANGER IS DRASTICALLY MODIFIED BY SULFHYDRYL BLOCKING-AGENTS - EVIDENCES THAT INTRACELLULAR CA(I)2+ REGULATORY AND TRANSPORT SITES ARE DIFFERENT

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 Ca(o)2+ or on the activatory external monovalent cation site. In contrast, when applied internally it drastically reduces the affinity of the Na+/Ca2+ exchanger towards Ca(i)2+ ions without affecting its maximal rate of transport; in the presence of MgATP the K0.5 for Ca(i)2+ activation of forward Na+/Ca2+ exchange increases from 1.5 muM to 95 muM; likewise the apparent affinity of the Ca(i)2+ stimulation of the reversal exchange decreases 100-fold. Interestingly, no effect of PCMBS was found on the interactions between Na(i)+ and Ca(i)2+ ions with the internal transport site(s) (inhibition of Na(o)+ and Ca(o)2+-dependent Ca2+ efflux by Na(i)+). On the other hand, Na(i)+ ions do not modify the interactions of Ca(i)2+ with that site. Two important characteristics of the Ca(i)2+ regulatory site are uncover in this work: (i) sulfhydryl groups are important in maintaining the integrity of the Ca2+ binding domain of the Ca(i)+ regulatory site and (ii) Na(i)+ and Ca(i)2+ regulatory, or Na(i)+ and Ca(i)+ transporting sites, are different entities.