A novel molecular determinant for cAMP-dependent regulation of the frog heart Na⁺-Ca²⁺ exchanger

Na⁺-Ca²⁺ exchanger is one of the major sarcolemmal Ca²⁺ transporters of cardiac myocytes. In frog ventricular myocytes the exchanger is regulated by isoproterenol via a β-adrenoreceptor/adenylate- cyclase/cAMP-dependent signaling pathway providing a molecular mechanism for the relaxant effect of the hormone. Here, we report on the presence of a novel exon of 27-base pair insertion, which generates a nucleotide binding motif (P-loop) in the frog cardiac Na⁺-Ca²⁺ exchanger. To examine the functional role of this motif, we constructed a full-length frog heart Na⁺- Ca²⁺ exchanger cDNA (fNCX1a) containing this exon. The functional expression of fNCX1a in oocytes showed characteristic voltage dependence, divalent (Ni²⁺, Cd²⁺) inhibition, and sensitivity to cAMP in a manner similar to that of native exchanger in frog myocytes. In oocytes expressing the dog heart NCX1 or the frog mutant (ΔfNCX1a) lacking the 9-amino acid exon, cAMP failed to regulate Na⁺-dependent Ca²⁺ uptake. We suggest that this motif is responsible for the observed cAMP-dependent functional differences between the frog and the mammalian hearts.