Mitochondrial sodium/calcium exchanger protein
calcium:sodium antiporter activity
Identification of residues that control Li<sup>+</sup> versus Na<sup>+</sup> dependent Ca<sup>2+</sup> exchange at the transport site of the mitochondrial NCLX.
<h4>Background</h4>The Na<sup>+</sup>/Ca<sup>2+</sup>/Li<sup>+</sup> exchanger (NCLX) is a member of the Na<sup>+</sup>/Ca<sup>2+</sup> exchanger family. NCLX is unique in its capacity to transport both Na<sup>+</sup> and Li<sup>+</sup>, unlike other members, which are Na<sup>+</sup> selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li<sup>+</sup> or Na<sup>+</sup> selective Ca<sup>2+</sup> transport and map their putative location on NCLX cation transport site.<h4>Method</h4>We combined molecular modeling to map transport site of NCLX with euryarchaeal H<sup>+</sup>/Ca<sup>2+</sup> exchanger, CAX_Af, and fluorescence analysis to monitor Li<sup>+</sup> versus Na<sup>+</sup> dependent mitochondrial Ca<sup>2+</sup> efflux of transport site mutants of NCLX in permeabilized cells.<h4>Result</h4>Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca<sup>2+</sup> exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li<sup>+</sup>/Ca<sup>2+</sup> exchange activity but a reduced Na<sup>+</sup>/Ca<sup>2+</sup> exchange activity. On the other hand, D471A showed dramatically reduced Li<sup>+</sup>/Ca<sup>2+</sup> exchange, but Na<sup>+</sup>/Ca<sup>2+</sup> exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca<sup>2+</sup> binding residues was required to completely abolish both Na<sup>+</sup>/Ca<sup>2+</sup> and Li<sup>+</sup>/Ca<sup>2+</sup> exchange activities.<h4>Conclusions</h4>We identified distinct Na<sup>+</sup> and Li<sup>+</sup> selective residues in the NCLX transport site. We propose that functional segregation in Li<sup>+</sup> and Na<sup>+</sup> sites reflects the functional properties of NCLX required for Ca<sup>2+</sup> exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane.<h4>General significance</h4>The results of this study provide functional insights into the unique Li<sup>+</sup> and Na<sup>+</sup> selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.