Chimeric analysis of Na+/Ca2+ exchangers NCX1 and NCX3 reveals structural domains important for differential sensitivity to external Ni2+ or Li+

Abstract

Externally applied Ni2+, which apparently competes with Ca2+ in all three isoforms of Na+/Ca2+ exchanger, inhibits exchange activity of NCX1 or NCX2 with a 10-fold higher affinity than that of NCX3, whereas stimulation of exchange by external Li+ is significantly greater in NCX2 and NCX3 than in NCX1 (Iwamoto, T., and Shigekawa, M. (1998) Am. J. Physiol. 275, C423- C430). Here we identified structural domains in the exchanger that confer differential sensitivity to Ni2+ or Li+ by measuring intracellular Na+- dependent 45Ca2+ uptake in CCL39 cells stably expressing NCX1/CX3 chimeras or mutants. We found that two segments in the exchanger corresponding mostly to the internal α-1 and α-2 repeats are individually responsible for the alteration of Ni2+ sensitivity, both together accounting for ~80% of the difference between NCX1 and NCX3. In contrast, the segment corresponding to the α-2 repeat fully accounts for the differential Li+ sensitivity between the isoforms. The Ni2+ sensitivity was mimicked, respectively, by simultaneous substitution of two amino acids in the α-1 repeat (N125G/T127I in NCX1 and G159N/I161T in NCX3) and substitution of one amino acid in the α-2 repeat (V82OA in NCX1 and ASO9V in NCX3). On the other hand, the Li+ sensitivity was mimicked by double substitution mutation in the α-2 repeat (V82OA/Q826V in NCX1 and ASO9V/V815Q in NCX3). Single substitution mutations at Asn125 and Val820 of NCX1 caused significant alterations in the interactions of the exchanger with Ca2+ and Ni2+, and Ni2+ and Li+, respectively, although the extent of alteration varied depending on the nature of side chains of substituted residues. Since the above four important residues are mostly in the putative loops of the α repeats, these regions might form an ion interaction domain in the exchanger.