Mutational analysis of potential pore-lining amino acids in TM IV of the Na(+)/H(+) exchanger.
The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). In this study we examined the effect of site-specific mutagenesis on the pore-lining amino acid Phe161 and effects of mutagenesis on the charged amino acids Asp159 and Asp172. There was no absolute requirement for a carboxyl side chain at amino acid Asp159 or Asp172. Mutation of Asp159 to Asn or Gln maintained or increased the activity of the protein. Similarly, for Asp172, substitution with a Gln residue maintained activity of the protein, even though substitution with an Asn residue was inhibitory. The Asp172Glu mutant possessed normal activity after correction for its aberrant expression and surface targeting. Replacement of Phe161 with a Leu demonstrated that it was not irreplaceable in NHE1 function. However, the mutation Phe161lys inhibited NHE1 function, while the Phe161Ala mutation caused altered NHE1 targeting and expression levels. Our results show that these three amino acids, while being important in NHE1 function, are not irreplaceable. This study demonstrates that multiple substitutions at a single amino acid residue may be necessary to get a clearer picture membrane protein function.
Slepkov
Emily
E
Department of Biochemistry, 347 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada.
Ding
Jie
J
Han
Jiayi
J
Fliegel
Larry
L
D000818:Animals; D016466:CHO Cells; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D018021:Lithium Chloride / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D012965:Sodium Chloride / Q000494:pharmacology; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship
D027682:Cation Transport Proteins; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride; D018021:Lithium Chloride