Ligand dependence of gating of single r-InsP₃R-3 channels. V_app = −40 mV. (A) Typical single-channel on-nucleus patch-clamp current traces of InsP₃R-3 channels in suboptimal (190 nM), optimal (2 µM), and inhibitory (23 µM) [Ca²⁺]_i in the presence of saturating (10 µM) [InsP₃], demonstrating biphasic [Ca²⁺]_i dependence of InsP₃R-3 channel activity. Arrow indicates closed-channel baseline current level for these and all subsequent current traces. (B) Typical single-channel on-nucleus patch-clamp current traces in the presence of subsaturating (3 µM) [InsP₃] showing that [InsP₃] reduction has little effect on channel activity at suboptimal (190 nM) [Ca²⁺]_i but increases channel sensitivity to Ca²⁺ inhibition, so channel activity is substantially decreased at [Ca²⁺]_i = 2 µM. (C) [Ca²⁺]_i dependence of mean channel P_o in various [InsP₃] as tabulated. Error bars show the SEM in this and all subsequent figures unless stated otherwise. The number of current traces analyzed for each data point is tabulated next to the data point in the same color. Curves are empirical biphasic Hill equation fits to mean P_o data points for various [InsP₃] with the same P_max, K_act, H_act, and H_inh. The purple inset shows the dependence of the K_inh on [InsP₃]. Error bars here show the estimates of fitting errors of K_inh derived from the biphasic Hill equation fits. The curve is the empirical simple Hill equation fit of the [InsP₃] dependence. (D and E) [Ca²⁺]_i dependence of mean open and closed durations of InsP₃R channel in various [InsP₃], derived from the same experimental data used in C. Data points in the same [InsP₃] are connected with lines for clearer presentation.