Abstract
Mammalian Na(+)/H(+) exchanger (NHE) isoforms are differentially sensitive to inhibition by several distinct classes of pharmacological agents, including amiloride- and benzoyl guanidinium-based derivatives. The determinants of drug sensitivity, however, are only partially understood. Earlier studies of the drug-sensitive NHE1 isoform have shown that residues within the fourth membrane-spanning helix (M4) (Phe(165), Phe(166), Leu(167), and Gly(178)) and a 66-amino acid segment encompassing M9 contribute significantly to drug recognition. In this report, we have identified two residues within M9, one highly conserved (Glu(350)) and the other non-conserved (Gly(356)), that are major determinants of drug sensitivity. In addition, residues in the second exomembrane loop between M3 and M4 (Gly(152), Phe(157), and Pro(158)) were also found to modestly influence drug sensitivity. A double substitution of crucial sites within M4 and M9 of NHE1 with the corresponding residues present in the drug-resistant NHE3 isoform (i.e. L167F/G356A) greatly reduced drug sensitivity in a cooperative manner to levels nearing that of wild type NHE3. The above mutations did not appreciably affect Na(o)(+) affinity but did markedly decrease the catalytic turnover of the transporter. These data suggest that specific sites encompassing M4 and M9 are critical determinants of both drug recognition and cation translocation.
Highlights
Properties, sensitivity to pharmacological antagonists, and responsiveness to various signaling pathways
The region surrounding M9 is of particular interest because it appears to account for large differences (2–3 orders of magnitude) in the differential drug sensitivity of NHE1 and NHE3 [16] and is proposed to form part of the pore-lining region with M4 [41]
The present study further demarcates this region by showing that Gly356 of NHE1, which is thought to reside within M9, is a crucial determinant of Na؉/H؉ exchanger (NHE) isoform-specific drug sensitivity
Summary
Properties, sensitivity to pharmacological antagonists, and responsiveness to various signaling pathways. Pharmacological analyses of chimeras of rat NHE1 and NHE3 defined a 66-amino acid segment encompassing M9 and its adjacent loops (residues 327–392 of rat NHE1) as a major determinant of the differential drug sensitivity between these two isoforms [16] Homologous substitution of this region between NHE1 and NHE3 caused a reciprocal change in their drug sensitivities by 1–3 orders of magnitude, with the greatest effects observed for the more NHE-selective drugs, EIPA and HOE694. A role for this region in drug recognition is further supported by observations of Wang et al [33], who serendipitously found that mutation of His349 in the putative M9 domain of human NHE1 produced either a modest 2-fold increase (H349Y or H349F) or 2-fold decrease (H349G or H349L) in amiloride sensitivity, other amino acid substitutions had no effect. This site is unlikely to account for the large changes observed for the NHE1/3 chimeras, it does implicate the involvement of other nearby sites as critical determinants of drug sensitivity
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