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(346)Asp mutant was more than 2000-fold more resistant to HOE694 and up to 300-fold more resistant to EIPA than wild-type NHE1, 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(346) did not change the sensitivity of mutated NHE1 proteins to inhibitors. We suggest there is a direct interaction of Glu(346) or involvement of Glu(346) in a coordination site with NHE inhibitors and with Na(+).
Noël
Josette
J
Groupe d'étude sur les protéines membranaires (GEPROM), département de physiologie, Université de Montréal, Montréal, Québec, Canada. josette.noel@umontreal.ca
Germain
David
D
Vadnais
Julie
J
D000584:Amiloride / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D004351:Drug Resistance / Q000235:genetics; D014644:Genetic Variation; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000737:chemistry; D011522:Protons; D012333:RNA, Messenger / Q000032:analysis; D012964:Sodium / Q000737:chemistry*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013450:Sulfones / Q000494:pharmacology
D006146:Guanidines; D011522:Protons; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C069139:growth factor-activatable Na-H exchanger NHE-1; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; D018698:Glutamic Acid; D000584:Amiloride; D012964:Sodium; D011188:Potassium