Calmodulin Interacts with the Sodium/Calcium Exchanger NCX1 to Regulate Activity

Changes in intracellular Ca(2+) concentrations ([Ca(2+)](i)) are an important signal for various physiological activities. The Na(+)/Ca(2+) exchangers (NCX) at the plasma membrane transport Ca(2+) into or out of the cell according to the electrochemical gradients of Na(+) and Ca(2+) to modulate [Ca(2+)](i) homeostasis. Calmodulin (CaM) senses [Ca(2+)](i) changes and relays Ca(2+) signals by binding to target proteins such as channels and transporters. However, it is not clear how calmodulin modulates NCX activity. Using CaM as a bait, we pulled down the intracellular loops subcloned from the NCX1 splice variants NCX1.1 and NCX1.3. This interaction requires both Ca(2+) and a putative CaM-binding segment (CaMS). To determine whether CaM modulates NCX activity, we co-expressed NCX1 splice variants with CaM or CaM(1234) (a Ca(2+)-binding deficient mutant) in HEK293T cells and measured the increase in [Ca(2+)](i) contributed by the influx of Ca(2+) through NCX. Deleting the CaMS from NCX1.1 and NCX1.3 attenuated exchange activity and decreased membrane localization. Without the mutually exclusive exon, the exchange activity was decreased and could be partially rescued by CaM(1234). Point-mutations at any of the 4 conserved a.a. residues in the CaMS had differential effects in NCX1.1 and NCX1.3. Mutating the first two conserved a.a. in NCX1.1 decreased exchange activity; mutating the 3(rd) or 4(th) conserved a.a. residues did not alter exchange activity, but CaM co-expression suppressed activity. Mutating the 2(nd) and 3(rd) conserved a.a. residues in NCX1.3 decreased exchange activity. Taken together, our results demonstrate that CaM senses changes in [Ca(2+)](i) and binds to the cytoplasmic loop of NCX1 to regulate exchange activity.