Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1.
NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.
Slepkov
Emily R
ER
Membrane Protein Research Group, Department of Biochemistry, Faculty of Medicine, Canadian Institute of Health Research, University of Alberta, 347 Medical Science Building, Edmonton, AB, Canada T6G 2H7.
Chow
Signy
S
Lemieux
M Joanne
MJ
Fliegel
Larry
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017354:Point Mutation; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection
D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D011392:Proline