Figure 5.
Screening for deviations from WT behavior of substrate activation kinetics at −50 mV using a two-point assay. (A) Two-point assay sensitivity for Pi activation (left) and Na+ activation (right). Continuous lines are the Pi activation index (ratio of response in 100 mM Na+ to 0.1 mM Pi and 1 mM Pi) as a function of apparent affinity for Pi (K m Pi) and Na+ activation index (ratio of response to 1 mM Pi in 50 mM Na+ and 100 mM Na+) as a function of apparent affinity for Na+ (K m Na), respectively. Indices were determined as a function of K m Pi or K m Na using the modified Hill equation to describe the electrogenic response to Pi, I Pi = I Pi max (S nH)/(S nH + K m SnH), where S is the concentration of the variable substrate, K m S is the apparent affinity constant, n H is the Hill coefficient (n H = 1.0 for Pi activation, n H = 2.5 for Na+-activation; Forster et al., 1998, 1999), and I Pi max is the Pi-dependent change in holding current at the saturating limit. In general, K m S and I Pi max can also depend on the concentration of the invariant substrate and holding potential (V h). Lower test concentration was chosen close to previously reported estimates for the WT apparent affinity, and upper test concentration was chosen close to the saturation concentration for Pi or maximum usable Na+ concentration, for the Pi and Na+ activation screens, respectively. For each case, the gray bar represents range of index values observed for WT-expressing oocytes (n = 9, three donor frogs). Vertical lines indicate typical K m Pi for WT (0.06 mM) and K m Na for WT (50 mM) at −50 mV, previously reported (e.g., Forster et al., 1998), respectively. (B) Pi activation index (top) and Na+ activation index (bottom), where each point is mean ± SEM for ≥4 oocytes. Gray bars indicate typical range observed for WT-expressing oocytes measured under the same experimental conditions. ND, not determined; −MTS, filled squares; +MTS, empty squares.