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.



from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2khtMzP
via IFTTT