Figure 1.

Stable transfection of COS-7 cells with wild-type or mutant B. marinus α1 subunits results in functional expression of ouabain-resistant Na,K-ATPase. ⁸⁶Rb uptake was measured under initial rates in COS-7 cells preincubated for 15 min at 37°C in the absence or in the presence of increasing concentrations of ouabain. The Na,K-ATPase-mediated ⁸⁶Rb uptake was obtained by subtraction of ouabain-insensitive ⁸⁶Rb uptake measured in the presence of 2.5 × 10⁻³ M ouabain. Results are expressed as fractional change (with respect to control value, i.e., absence of ouabain) and are means ± SE from three or four independent experiments. (A) The ouabain sensitivity of the endogenous Na,K-ATPase of nontransfected (NT) COS-7 cells was monophasic (IC₅₀ = 2 × 10⁻⁷ M) and best described by Eq. 1 (r² = 0.97; see MATERIALS AND METHODS). (B–D) COS-7 cells transfected with the wild-type (B; WT), T15A/S16A mutant (C), or S16D mutant (D) Bufo α1 subunit cDNAs exhibited a bimodal ouabain inhibition pattern best described by Eq. 2 (r² = 0.95–0.98; see MATERIALS AND METHODS). The Na,K-ATPase populations with high ouabain sensitivity exhibited IC₅₀ values between 2 × 10⁻⁸ and 4 × 10⁻⁷ M, and the Na, K-ATPase populations with lower ouabain sensitivity displayed IC₅₀ values between 1 and 3.5 × 10⁻⁵ M.