Τετάρτη 25 Ιανουαρίου 2017

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

Publication date: Available online 24 January 2017
Source:Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
Author(s): Soumitra Roy, Kuntal Dey, Michal Hershfinkel, Ehud Ohana, Israel Sekler
BackgroundThe 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 to identify NCLX residues that confer Li+ or Na+ selective Ca2+ transport and map their putative location on NCLX cation transport site.MethodWe 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.ResultMutation 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.ConclusionsWe 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 SignificanceThe results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger.



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