A Glu³²⁹→ Gln variant of the α‐subunit of the rat kidney Na⁺K⁺‐ATPase can sustain active transport of Na⁺ and K⁺ and Na⁺K⁺‐activated ATP hydrolysis with normal turnover number

Abstract

An allelic variant of the ouabain-in~nsitive rat kidney Na',K+-ATPase a,-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na',K'-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue G~u'*~ previously suggested as a likely donator of oxygen ligands for Na+ and K' binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K'-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu'*9+Gln variant, whose cDNA was shown by polymerase chain reaction ~pIi~cation to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu3"-+Gln variant did not differ si~ifi~ntly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Gh_?+Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.

Amino acid substitutions in the rat Na+, K(+)‐ATPase alpha 2‐subunit alter the cation regulation of pump current expressed in HeLa cells.

“…The Kd values for both E327Q and D925L were higher, namely 7-7 and 6-4 mm, respectively. The 6-4-fold decrease in affinity observed for E327Q is similar to that observed in biochemical studies previously reported (Vilsen, 1993). The Kd values calculated from ATPase assays were 1P0, 3*7 and 1-9 for the rat a2*, E327Q and D925L proteins, respectively.…”

A Conformation-specific Interhelical Salt Bridge in the K+ Binding Site of Gastric H,K-ATPase

“…The experiments of Koster et al (32), which show that the properties of the E786K mutant in Na,K-ATPase are rather similar to that of the wild type, do not fit in the model for Na,K-ATPase (27). In the latter model Glu 334 does not play a direct role, but mutagenesis experiments with Na,K-ATPase suggest that this residue is directly involved in K ϩ -binding (30,(33)(34)(35).…”

Mutation of Amino Acid Residues in the Putative Transmembrane Segments of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

“…The COOH-terminal amino acids, PEGLLA, are involved in ion binding. Mutations at the proline, glutamate, or the second leucine residue have been shown to alter the affinities for K ϩ and Na ϩ (30). In an analogous region of the sarcoplasmic reticular Ca 2ϩ pump, the residues EGL have been shown to be involved in Ca 2ϩ binding and transport and in the conformational changes associated with Ca 2ϩ transport (31).…”

“…Glu-199 is conserved among the three NCX-type exchangers, and the analagous glutamate in the Na ϩ , K ϩ , and Ca 2ϩ pumps is involved in ion binding and translocation (30,31). Conservative mutations at Glu-199 to either glutamine or aspartate resulted in non-functional exchangers.…”