Background The Na+/Ca2 +/Li+ exchanger (NCLX) is a member of the Na+/Ca2 + 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 Ca2 + 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+/Ca2 + exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2 + 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 Ca2 + exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2 + exchange activity but a reduced Na+/Ca2 + exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2 + exchange, but Na+/Ca2 + exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2 + binding residues was required to completely abolish both Na+/Ca2 + and Li+/Ca2 + 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 Ca2 + 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.
|Number of pages||12|
|Journal||Biochimica et Biophysica Acta - Molecular Cell Research|
|State||Published - 1 Jun 2017|
- 3D modelling
- Li/Ca exchange
- Na/Ca exchange
- Permeabilized cells