Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Abstract

Site-directed mutagenesis was used to identify residues responsible for the > 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase al and at2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat at2 subunit with the corresponding residues from the rat al subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabainsensitive primate cells. Either of two single substitutions introduced into the rat ai2 subunit cDNA (Leu-111-*Arg or Asn-122-*Asp) conferred partial resistance (-10 ,uM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat al cDNA (-500 ,uM ouabain) and the rat a2 cDNA (-0.2 ,iM ouabain). A double substitution of the rat a2 cDNA (Leu-111--*Arg and Asn-122-Asp) conferred a resistance level equivalent to that obtained with rat al. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the ends of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.

Ouabain Interactions with the H5-H6 Hairpin of the Na,K-ATPase Reveal a Possible Inhibition Mechanism via the Cation Binding Domain

“…Identification of Ouabain Sensitivity-The cardiac glycoside binding site of the Na,K-ATPase has been the target of numerous site-directed mutagenesis studies (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)24). Based on the extracellular action of the drug, these studies have focused on residues in the extracellular or transmembrane domains which vary in a species-specific manner (i.e.…”

“…In these mutagenesis studies, specific amino acid substitutions were introduced into a ouabain-sensitive ␣ isoform by site-directed mutagenesis, and the altered cDNAs were transfected into cells carrying an endogenous ouabain-sensitive ␣1 isoform. Amino acid substitutions that prevent inhibition by ouabain conferred ouabain resistance to the sensitive cells (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Site-specific integration by adeno-associated virus is directed by a cellular DNA sequence.

“…p220.2 contains the EBV latent origin of replication oriP, the EBNA-1 gene, whose product transregulates oriP, a hygromycin-resistance gene for the selection of C17 cells propagating this shuttle vector, and the pBR322 replication origin and ampicillin-resistance gene for selection in E. coli (14,15). The C17 derivative of the adenovirus 5-transformed human 293 cell line expresses EBNA-1 constitutively, which may facilitate the propagation of p220.2 as an extrachromosomal nuclear episome (16,22). C17 cells carrying p220.2/AAVS1 episomes were infected with AAV at various cell passage levels.…”

Computational Study of Bufadienolides from Indonesia’s Kalanchoe Pinnata as Na+/K+-ATPase Inhibitor for Anticancer Agent

“…In contrast, Kamano et al suggested that the 11-OH was not an important substituent for ligand binding. 29 Interestingly, Supratman et al found that the 11-OH group decreased the cytotoxicity of bufadienolides. 15 Thus, it is indicated that the formation of hydrogen bonds with Ile315 (in C2) and Asn122 (in C1) would increase the ligand's selectivity to NaK-ATPase.…”