title: Corroboration of Dahl S Q276L alpha1Na,K-ATPase protein sequence: impact on affinities for ligands and on E1 conformation.
abstract: OBJECTIVE
  Multifactorial analyses support the hypothesis that alpha1Na,K-ATPase is a hypertension susceptibility gene in Dahl S rats. However, two studies report non-detection of the A1079T transversion underlying the Q276L substitution in Dahl S alpha1Na,K-ATPase questioning the validity of ATP1A1 as a hypertension susceptibility gene. To resolve this discordance, we investigated the issue at the protein level.
  
  
  METHODS
  We employed protein blot analysis using Q276L- and Q276-specific; antipeptide-specific antibodies; tested differential chymotrypsin cleavage efficiency, measured differential Na and K affinities of alpha1Na,K-ATPases in Dahl S and Dahl R renal membranes and determined amino acid sequences of purified Dahl S alpha1Na,K-ATPase chymotryptic-digest peptides.
  
  
  RESULTS
  We detected Q276L variant protein in Dahl S rats; and Q276 wild-type variant in Dahl R, spontaneously hypertensive (SHR), Lewis and Wistar-Kyoto (WKY) rat kidney membranes. Q276L variant exhibits less chymotrypsin cleavage efficiency than the Q276 wild-type variant, consistent with the substitution of hydrophobic L for hydrophilic Q. Kinetic studies of kidney membranes detect increased Na affinity and decreased K affinity in renal Dahl S alpha1Na,K-ATPase compared with Dahl R. Protein sequencing of high pressure liquid chromatography (HPLC)-purified chymotrypsin digested 77 kDa peptide confirms Q276L substitution in the Dahl S alpha1Na,K-ATPase.
  
  
  CONCLUSIONS
  Data demonstrate the existence and functional significance of the Q276L variant in Dahl S rats.
authors.affiliation: Section of Molecular Medicine, Department of Medicine, Boston University School of Medicine, 700 Albany Street, W-609, Boston, Massachusetts 02118, USA.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kaneko
authors.forename: Jean-Francois
authors.initials: JF
authors.lastname: Cloix
authors.forename: Victoria Lm
authors.initials: VL
authors.lastname: Herrera
authors.forename: Nelson
authors.initials: N
authors.lastname: Ruiz-Opazo
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D006973:Hypertension / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008024:Ligands; D051381:Rats; D020303:Rats, Inbred Dahl; D011917:Rats, Inbred Lew; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D020539:Sequence Analysis, Protein; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*
chemical_list: D008024:Ligands; D000254:Sodium-Potassium-Exchanging ATPase