A Novel Topology Model of the Human Na+/H+ Exchanger Isoform 1

Abstract

The membrane topology of the human Na ؉ /H ؉ exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethylmethanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.

Mutations of Arg440 and Gly455/Gly456 Oppositely Change pH Sensing of Na+/H+ Exchanger 1

“…Construction of Na ϩ /H ϩ Exchanger Mutants-A plasmid carrying cDNA coding for the Na ϩ /H ϩ exchanger (NHE1 human isoform) containing some unique restriction sites cloned into mammalian expression vector pECE was described previously (18). A cDNA construct for Cys-less NHE1 devoid of all endogenous cysteine residues was also described previously (20). For construction of point mutant cDNAs, we used a PCR-based strategy as described previously (20), using two template plasmids coding for the wild-type or Cys-less NHE1.…”

“…A cDNA construct for Cys-less NHE1 devoid of all endogenous cysteine residues was also described previously (20). For construction of point mutant cDNAs, we used a PCR-based strategy as described previously (20), using two template plasmids coding for the wild-type or Cys-less NHE1. Briefly, (20).…”

“…Based on the accessibility of introduced cysteine residues to sulfhydryl reagents, we have recently presented a new membrane topology model of NHE1 in which the exchanger consists of 12 transmembrane segments with N-and C-tails located in the cytosol (20), although a previous study reported the N-tail to be cleaved as a signal peptide during the exchanger biogenesis (21). In a subsequent cysteine-scanning mutagenesis study, we found that mutation of Tyr 454 or Arg 458 in a new transmembrane domain 11 (TM11) impaired the plasma membrane expression of NHE1 (22), suggesting the importance of these residues for proper folding of NHE1.…”

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

“…A, topological model of the NHE1 isoform of the Na ϩ H ϩ exchanger. The orientation of TM segments I-XII of the NHE1 isoform of the Na ϩ /H ϩ exchanger is illustrated (6). B, crude model of amino acids present in TM IV.…”

“…1). The N-terminal membrane domain is responsible for ion movement, and it is reported to have 12 transmembrane (TM) segments and 3 membrane-associated segments (6). Transmembrane segment four (TM IV; residues 155-177) has been implicated in the ion transport and inhibitor binding properties of NHE1 (7)(8)(9).…”