Methods to assess gap junction permeability. (A) Dual whole-cell and perforated-patch clamp has been used to assess gap junction conductance and to deliver the fluorescent probe to the donor cell (rightmost cell) of a pair. (B) LY flux between HeLaCx40G38D cells. Fluorescence emission is monitored over time (1-, 5-, and 12-min records shown) after an LY injection to the rightmost cell of a pair. Bar, 10 µm. (C) Plot of fluorescence intensity versus time for an injected cell (●) and a recipient cell (○). Shaded area indicates the 12-min time point at which the ratio of the recipient cell’s fluorescence intensity to the source cell’s fluorescence intensity was calculated. (D; top) I_j evoked by V_j pulses (g_j = 18nS). (Bottom) The application of 100% CO₂ produced a progressive decrease in I_j. (E) Summary plots of the relative fluorescence intensity versus g_j for LY in cells expressing Cx40 mutants: M163V (yellow circles), G38D (green circles), A96S (red circles), WT hCx40 (white circles), and WT rCx40 (black circles). Each data point corresponds to the relative fluorescence intensity (the ratio of the fluorescence intensity of the recipient cell over that of the injected cell) obtained at the 12-min time point after dye injection. Solid lines represent the first-order regressions, and dashed lines represent 95% confidence intervals.