Identification of residues that control Li⁺ versus Na⁺ dependent Ca^{2 +} exchange at the transport site of the mitochondrial NCLX

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