PB cassette substitution masks con action by raising gating efficacy: refutation of necessity hypothesis. (A) Dose–response relations of Construct 3 activated with cGMP (down-triangles) or cAMP (up-triangles). Points show means and error bars show SD (n ≥ 3 for each point); points are joined by straight lines without fitting. To plot P_o values, conductances were normalized to the steady-state conductance measured at 3 mM cGMP (solid down-triangle) in the same patch. Then, P_o at 3 mM cGMP was fixed as 0.73 based on Ni²⁺ potentiation (see Materials and methods). For comparison, gray curves show dose–response relations for Construct 2 activated by cGMP (solid) or cAMP (dashed); these were previously reported on a relative scale without conversion to P_o values (Chan and Young, 2009), and new Ni²⁺ potentiation experiments in this study fix the P_o at 3 mM cGMP as 0.18. All measures for both constructs in this plot were collected after completion of spontaneous run-up. (B) Example macroscopic current trace from one patch of Construct 3 before and after completion of run-up, tracking progressive increase in pro-action efficacy and masking of con action. P_o scale at right is based on fixing P_o = 0.73 for 3 mM cGMP after run-up. Arrows and asterisks, respectively, mark steady-state and spike currents as in Fig. 1. For this example, P_steady/P_spike = 0.80 before run-up and 0.95 after run-up.