Glutamate 346 of human Na+-H+ exchanger NHE1 is crucial for modulating both the affinity for Na+ and the interaction with amiloride derivatives.

A NHE1 variant that exhibits very high resistance to (3-methyl sulfonyl-4-piperidinobenzoyl) guanidine methane sulfonate (HOE694), a potent inhibitor of Na + -H + exchangers, was selected and characterized. Sequencing of the coding region corresponding to the N-terminal domain of this variant revealed the presence of only one mutation located within membrane-spanning segment 9 (M9). This base pair change replaces a glutamate (Glu) with an aspartate (Asp). We reproduced this amino acid change in wild-type NHE1 and found that this mutation alone is responsible for the huge decrease in sensitivity to the HOE694 compound and to ethylisopropylamiloride (EIPA). We found that the NHE1-Glu 3 4 6 Asp mutant was more than 2000-fold more resistant to HOE694 and up to 300-fold more resistant to EIPA than wild-type NHEI, with the size, rather than the charge, of the amino acid in position 346 having the greatest effect. Interestingly, its affinity for Na + was at least 4-fold lower than that of wild-type NHE1. Mutation of amino acids in the vicinity of Glu 3 4 6 did not change the sensitivity of mutated NHE1 proteins to inhibitors. We suggest there is a direct interaction of Glu 3 4 6 or involvement of Glu 3 4 6 in a coordination site with NHE inhibitors and with Na + .