Abstract
The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H(+) in exchange for one extracellular Na(+). We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys(447)-Lys(472)) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp(447)-Tyr(454) and Phe(460)-Lys(471) at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly(455)-Gly(459). TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na(+)/H(+) exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu(465) contributing to the pore.
Highlights
Prolines 167 and 168 of TM IV were critical to Naϩ/Hϩ exchanger isoform 1 (NHE1) function (12) and cysteinescanning mutagenesis was used to show that Phe161 is a pore lining residue critical to transport
We found that TM IV of NHE1 has a similar structure and function to that of TM IV of NhaA (2, 13), leaving open the possibility that TM XI of NHE1 is similar in structure and function to TM XI of NhaA
In this study we examined the structural and functional characteristics of TM XI of the NHE1 isoform of the Naϩ/Hϩ exchanger
Summary
5Ј-GTATCGTGAAGCTTACCCCCAAGGACTGCTTCATCATCGCC-3Ј 5Ј-CCCCCAAGGACCAATGCATCATCGCCTATGG-3Ј 5Ј-CCCAAGGACCAGTTCTGCATCGCATATGGGGGCCTGCGAG-3Ј 5Ј-GGACCAGTTCATCTGCGCATATGGGGGCCTGC-3Ј 5Ј-CCAGTTCATCATCTGCTATGGAGGCCTGCGAGGGGC-3Ј 5Ј-CAGTTCATCATCGCATGCGGGGGCCTGCGAGG-3Ј 5Ј-CATCATCGCCTATTGCGGCTTAAGAGGGGCCATCGCC-3Ј 5Ј-CATCGCCTATGGGTGCTTAAGAGGGGCCATCGCC-3Ј 5Ј-GCCTATGGGGGCTGCCGCGGGGCCATCGCCTTC-3Ј 5Ј-CATCGCCTATGGAGGCCTGTGTGGGGCCATCGCC-3Ј 5Ј-GGGGGCCTGCGATGCGCAATCGCCTTCTCTC-3Ј 5Ј-GGGGCCTGCGAGGATGCATCGCCTTCTCTC-3Ј 5Ј-GGCCTGCGAGGGGCATGCGCCTTCTCTCTGGG-3Ј 5Ј-CTGCGAGGGGCCATATGCTTCTCTCTGGGC-3Ј 5Ј-GAGGGGCCATCGCATGCTCTCTGGGCTACC-3Ј 5Ј-GGCCATCGCCTTCTGCCTAGGCTACCTCCTGG-3Ј 5Ј-CATCGCCTTCTCTTGCGGGTACCTCCTGGACAAG-3Ј 5Ј-CGCCTTCTCTCTGTGCTACCTTCTAGACAAGAAGCACTTC-3Ј 5Ј-CCTTCTCTCTGGGCTGTCTACTGGACAAGAAGCAC-3Ј 5Ј-CTCTCTGGGCTACTGTCTAGACAAGAAGCACTTC-3Ј 5Ј-GCCTTCTCTCTGGGATATCTCTGCGACAAGAAGCACTTC-3Ј 5Ј-GGGCTACCTCCTGTGCAAAAAGCACTTCCCCATG-3Ј. Ture of the bacterial Naϩ/Hϩ exchanger NhaA has been elucidated Both TM IV and TM XI play a critical role forming an assembly that cross, with each being a helix, an extended polypeptide and a short helix (19). Evidence has suggested that TM segments of membrane proteins possess all the structural information required to form their higher order structures in their amino acid sequence (20). This has been demonstrated in earlier studies on membrane protein segments such as the cystic fibrosis transmembrane conductance regulator (21), a fungal G-proteincoupled receptor (22), bacteriorhodopsin (23, 24), and rhodopsin (25), where it was shown that isolated TM segments from membrane proteins had structures in good agreement with the segments of the entire protein. The structure of TM XI was similar to the x-ray structure of TM XI of NhaA
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