Second mutations rescue point mutant of the Na⁺/H⁺ exchanger NHE1 showing defective surface expression

Abstract

We studied the effect of point mutation within the putative 11th transmembrane domain (TM11) of the Na + /H + exchanger NHE1 on the plasma membrane expression. Of the 19 mutants tested, two mutants (Tyr454 or Arg458 replaced by Cys) were retained in the endoplasmic reticulum. Interestingly, Y454C was expressed on the cell surface when one of the endogenous cysteine residues at position 8, 133, 421, or 477 was substituted with alanine. Random mutagenesis at Cys8 and its surrounding residues in the cytosolic N-tail revealed that replacement of Cys8 with Ala was the only identified single residue mutation that rescued Y454C. These results suggest that the abnormal conformation of the region of TM11 containing the Y454C mutation is compensated by the second mutation within other domains such as the N-tail. This approach may provide evidence for the interdomain interaction in NHE1. ß

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

“…Amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was a gift from the New Drug Research Laboratories of Kanebo, Ltd. (Osaka, Japan). 22 NaCl was purchased from PerkinElmer Life Sciences. An NHE1-specific polyclonal antibody (RP-cd) was raised as described previously (8).…”

“…Measurement of 22 Na ϩ Uptake-22 Na ϩ uptake activity and its pH i dependence were measured by the K ϩ /nigericin pH i clamp method (19). Serum-depleted cells in 24-well dishes were preincubated for 30 min at 37°C in Na ϩ -free choline chloride/KCl medium containing 20 mM Hepes/Tris (pH 7.4), 1.2-140 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM glucose, and 5 M nigericin.…”

“…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. Furthermore, our preliminary experiments suggested that some residues in TM11 regulate pH i sensitivity.…”

“…Substitution of Arg 440 in IL5 with other residues except positively charged Lys caused a large shift in pH i dependence of 22 Na ؉ uptake to an acidic side, whereas substitution of Gly 455 or Gly 456 within the highly conserved glycine-rich sequence of TM11 shifted pH i dependence to an alkaline side. The observed alkaline shift was larger with substitution of Gly 455 with residues with increasing sizes, suggesting the involvement of the steric effect.…”

“…The observed alkaline shift was larger with substitution of Gly 455 with residues with increasing sizes, suggesting the involvement of the steric effect. Interestingly, mutation of Arg 440 (R440D) abolished the ATP depletion-induced acidic shift in pH i dependence of 22 Na ؉ uptake as well as the cytoplasmic alkalinization induced by various extracellular stimuli, whereas with that of Gly 455 (G455Q) these functions were preserved. These mutant exchangers did not alter apparent affinities for extracellular transport substrates Na ؉ and H ؉ and the inhibitor 5-(N-ethyl-N-isopropyl)amiloride.…”