Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance.

Abstract

The cardiac glycoside ouabain inhibits Na,K‐ATPase by binding to the alpha subunit. In a highly ouabain resistant clone from the MDCK cell line, we have found two alleles of the alpha subunit in which the cysteine, present in the wild‐type first transmembrane segment, is replaced by a tyrosine (Y) or a phenylalanine (F). We have studied the kinetics of ouabain inhibition by measuring the current generated by the Na,K‐pump in Xenopus oocytes injected with wild‐type and mutated alpha 1 and wild‐type beta 1 subunit cRNAs. When these mutations, alpha 1C113Y and alpha 1C113F [according to the published sequence [Verrey et al. (1989) Am. J. Physiol., 256, F1034] were introduced in the alpha 1 subunit of the Na,K‐ATPase from Xenopus laevis, the inhibition constant (Ki) of ouabain increased greater than 1000‐fold compared with wild‐type. A more conservative mutation, serine alpha 1C113S did not change the Ki. We observed that the decreased affinity for ouabain was mainly due to a faster dissociation, but probably also to a slower association. Thus we propose that an amino acid residue of the first transmembrane segment located deep in the plasma membrane participates in the structure and the function of the ouabain binding site.

Molecular basis for the insensitivity of the Monarch (Danaus plexippus) to cardiac glycosides

“…In rats, a ouabnin-insensitive enzyme has been reported in which the substitution of orginine for a glutamine at position 111, and aspartic acid for an asparagine at position 122, is responsible, as compared to sensitive Na+,K*-ATPases from man or sheep [12]. Recently, evidence was presented that, in addition, also the first transmembrane segment of 'Na',K*-ATPase confers ouabain resistance in MDCK canine cells [23].…”

“…It cannot be ruled out that other mutations, e.g. in the first transmembrane segment [23] or in other extracellular loop regions, may be involved additionally in ouabain insensitivity.…”

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

“…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).…”

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

“…For example, the effects of amino acid substitutions on Ip have been studied using a transient Xenopus oocyte expression system in which cRNAs encoding mutant a-subunit and fl-subunit were co-injected and the endogenous oocyte Ip was inhibited by ouabain (Canessa, Horisberger, Louvard & Rossier, 1992). Although useful, the oocyte system has some limitations for electrophysiological analysis of Ip due to the transient nature of the expression and the extensive infoldings of the oocyte membrane, which greatly decrease the speed of voltage-clamp control and may impair ion exchange (Yatani, Wakamori, Mikala & Bahinski, 1993).…”