Identification of a peptide inhibitor of the cardiac sarcolemmal Na⁺-Ca²⁺ exchanger

The deduced amino acid sequence of the cardiac sarcolemmal Na⁺-Ca²⁺ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na⁺-Ca²⁺ exchange activity. The peptide inhibits the Na(i)⁺-dependent Ca²⁺ uptake (KI ~ 1.5 μM) and the Na(o)⁺-dependent Ca²⁺ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na⁺ and Ca²⁺. The peptide is also a potent inhibitor (K(I) ~ 0.1 μM) of the Na⁺-Ca²⁺ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na⁺-Ca²⁺ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.