InsP₃R channel P_o is independent of cytoplasmic Ca²⁺-buffering conditions in the absence of Ca²⁺ flux through the channel. (A–C) Typical single-channel on-nucleus patch-clamp current traces with [Ca²⁺]_i in pipette solutions buffered to 2 µM by 5 mM diBrBAPTA (A), 0.5 mM diBrBAPTA (B), or 0.1 mM HEDTA (C). [InsP₃] = 3 µM and V_app = −40 mV. Bath solution contained [Ca²⁺]_ER = 70 nM. (D) Mean InsP₃R channel P_o for [InsP₃] = 3 µM and [Ca²⁺]_i = 2 µM in various cytoplasmic Ca²⁺-buffering conditions for on-nucleus (closed bars) and excised lum-out (open bars) patch-clamp experiments. Excised lum-out patches were perfused with solution containing [Ca²⁺]_ER = 70 nM. H stands for HEDTA, and dB stands for diBrBAPTA. No statistically significant difference exists between P_o in any two of the three Ca²⁺-buffering conditions plotted for on-nucleus or lum-out experiments (ANOVA).