Identification of residues that control Li+ versus Na+ dependent Ca2+ exchange at the transport site of the mitochondrial NCLX

Abstract

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.

Keywords: 3D modelling; Li(+)/Ca(2+) exchange; NCLX; Na(+)/Ca(2+) exchange; Permeabilized cells.