Abstract
The Na(+)/H(+) exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H(+) for one extracellular Na(+). It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251-273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na(+)/H(+) exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly alpha-helical, with a break in the helix at the functionally critical residues Gly(261)-Glu(262). The relative positions and orientations of the N- and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition.
Highlights
The mammalian Naϩ/Hϩ exchanger isoform 1 (NHE1)6 is a ubiquitous integral membrane protein mediating removal of a single intracellular proton in exchange for one extracellular sodium ion [1]
We have shown that TM VII is critical for the function of the NHE1 isoform of the Naϩ/Hϩ exchanger [14]
Site-directed Mutagenesis—To examine and characterize critical amino acids of TM VII of the Naϩ/Hϩ exchanger, mutations were made to an expression plasmid containing a hemagglutinin (HA)-tagged human NHE1 isoform of the Naϩ/Hϩ exchanger
Summary
Materials—EMD87580 was a generous gift of Dr N. PWO DNA polymerase was from Roche Applied Science, and LipofectamineTM 2000 reagent was from Invitrogen. Site-directed Mutagenesis—To examine and characterize critical amino acids of TM VII of the Naϩ/Hϩ exchanger, mutations were made to an expression plasmid containing a hemagglutinin (HA)-tagged human NHE1 isoform of the Naϩ/Hϩ exchanger. One series of mutants was made in which all the residues of TM VII were mutated to alanine. A second series was for insertional mutagenesis, whereby alanine residues were inserted at critical locations between residues of TM VII in the wild type pYN4ϩ. Transfection with LipofectamineTM 2000 reagent (Invitrogen) was used to make stable cell lines of all mutants as described earlier [12]. Mutated amino acid residues are indicated using single letter notation, and new restriction sites are underlined. Amino acids flanking the insert are indicated where insertional mutagenesis occurred.
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