(A) Cartoon and Markov models of potential modulator binding schemes. Model 1 possesses mutually exclusive modulator binding whereas Model 2 can bind either or both modulators. Ligand-dependent transitions are denoted by [A] (agonist), [N] (NAM), [P] (PAM). (B) Simulated concentration-response curves of hypothetical modulator actions on an agonist-saturated response of Model 1 and 2 (NAM EC500.89 µM, PAM EC500.85 µM). (C) Simulation of co-administered hypothetical modulators for Model 1 (left) and Model 2 (right) to an agonist-saturated response. Responses to NAM concentrations (0.03, 0.1, 0.3, 1, 3, 10, 30, 100 µM) were simulated in the presence of fixed concentrations of PAM (0, 0.3, 1, 3, 10, 30 µM). (D) Simulated concentration-response data fit with the Hill equation for Model 1 (left) and Model 2 (right). (F) Simulated responses for co-application of multiple modulators using Model 1 and Model 2 (left and right, respectively). The current response to saturating agonist concentration was simulated in the presence of eight PAM concentration responses (0.03, 0.1, 0.3, 1, 3, 10, 30, 100 µM) and fixed concentrations of NAM (0, 0.3, 1, 3, 10, 30 µM). (G) The steady-state responses from each condition was plotted as a function of concentration and fit with the Hill equation. Rates used are (concentration dependent rates in µM s−1) b+=10.4 [A]*s−1, b-=73 s−1, k+=501.6 s−1, k-=790 s−1, n+=0.786 [N]*s−1, n-=0.393 s−1, p+=0.786 [P]*s−1, p-=0.786 s−1, kn+=25.08 s−1, kn-=790 s−1, kp+=1003.2 s−1, kp-=790 s−1, kpn+=50.16 s−1, kpn-=790 s−1.