Scientific Contributions Confirmation of Mutant ctx Na,K-ATPase Gene and Transcript in Dahl Salt-Sensitive/JR Rats

As the sole renal Na,K-ATPase isozyme, the a, Na,K-ATPase accounts for all active transport of Na throughout the nephron. This role in renal Na reabsorption and the primacy of the kidney in hypertension pathogenesis make it a logical candidate gene for salt-sensitive genetic hypertension. An adenine (A)—>thymine (T) transversion, resulting in the substitution of glutamine with leucine and associated with decreased net *Rb (K) influx, was identified in Dahl saltsensitive/JR rat kidney a, Na,K-ATPase cDNA. However, because a Taq polymerase chain reaction amplification-based reanalysis did not detect the mutant T but rather only the wild-type A a, Na,K-ATPase allele in Dahl salt-sensitive rat genomic DNA, we reexamined a, Na,K-ATPase sequences using Taq polymerase error-independent amplification-based analyses of genomic DNA (by polymerase allele-specific amplification and ligase chain reaction analysis) and kidney RNA (by mRNA-specific thermostable reverse transcriptase-polymerase chain reaction analysis). We also performed modified 3' mismatched correction analysis of genomic DNA using an The Na,K-ATPase is the integral plasma membrane protein, composed of an aand £-subunit, that maintains the Na-K electrochemical gradient in all mammalian cells. The a-subunit is the catalytic subunit to which Na,K-ATPase enzymatic properties have been mapped; the /3-subunit is necessary for functional assembly. Of the three a-subunit isoforms identified to date, ax, a2, and a3, 3 the investigation of the molecular genetic involvement of a, Na,KATPase in genetic hypertension is the most logical based on the following independent facts. First, the ct\ isoform is the sole a isoform in rat kidney and therefore accounts for all active transport of Na across the basolateral renal epithelial membrane of tubular segments throughout the nephron. It also provides the "downhill" Na gradient allowing the transport of Na across the apical renal epithelial membrane through various Na-coupled transporters and Na channels. Second, the kidney plays a pivotal pathogenic role in the Dahl genetic rat model of salt-sensitive hypertension primordial to the vasculature and neural cardiovascular Received December 21, 1993; accepted in revised form June 20, 1994. From the Section of Molecular Genetics, Whitaker Cardiovascular Institute, Boston (Mass) University School of Medicine (N.R.-O., V.L.M.H.); the Department of Microbiology, Cornell University School of Medicine, New York, NY (F.B.); and the Department of Pediatrics, Wakayama (Japan) Medical College (K.H.). Correspondence to V.L.M. Herrera, Whitaker Cardiovascular Institute, Boston University School of Medicine, 80 East Concord St, Boston, MA 02118. © 1994 American Heart Association, Inc. exonuclease-positive thermostable DNA polymerase. All the confirmatory test results were concordant, confirming the A-+T transversion in the Dahl salt-sensitive ax Na,KATPase allele and its transcript, as well as the wild-type A sequence in the Dahl salt-resistant a, Na,K-ATPase allele and its transcript. Documentation of a consistent Taq polymerase error that selectively substituted A at T (sense strand) was obtained from Taq polymerase chain reaction amplification and subsequent cycle sequencing of reconfirmed known Dahl salt-sensitive/JR rat mutant T a, cDNA M13 subclones. This Taq polymerase error results in the reversion of mutant sequence back to the wild-type a, Na,K-ATPase sequence. This identifies a siteand nucleotide-specific Taq polymerase misincorporation, suggesting that a structural basis might underlie a predisposition to nonrandom Taq polymerase errors. (Hypertension. 1994^4:260-270.)