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. DESIGN AND 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.
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. DESIGN AND 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: Victoria L Herrera; Khristine A Pasion; Ann Marie Moran; Roberta Zaninello; Maria Francesca Ortu; Giovanni Fresu; Daniela Antonella Piras; Giuseppe Argiolas; Chiara Troffa; Valeria Glorioso; Wanda Masala; Nicola Glorioso; Nelson Ruiz-Opazo Journal: PLoS One Date: 2015-01-23 Impact factor: 3.240