Fig. 3.

Functional expression of pannexins in single Xenopus oocytes. (A) Whole-cell membrane currents (I_m) were measured from single oocytes coinjected with pannexin RNAs and an oligonucleotide antisense to Xenopus Cx38 (see Materials and Methods). Cells were initially clamped at a membrane potential (V_m) of -40 mV, and depolarizing steps lasting 2 sec were applied in 10-mV increments up to +60 mV (bottom traces). For clarity, representative traces are shown only in 20-mV increments. (B) Current-voltage relationships were determined for oocytes injected with either antisense oligonucleotides (blue) or Px1 (black), Px2 (red), and Px3 (green) RNAs plus antisense. Peak current values above holding currents (ΔI_m) were calculated and plotted as a function of V_m. Mean values from Px1-injected cells were significantly different (P < 0.01) from those of control oocytes starting at a V_m of -10 mV. For Px1 steady-state currents (open circles), values recorded for 20 msec at the end of the pulse were averaged and plotted as above. Results are shown as mean ± SEM from at least eight independent experiments. Antisense, n = 45; Px1, n = 80; Px2, n = 46; Px3, n = 41. (C-F) Functional interaction of Px1 and Px2 proteins. Antisense-treated oocytes were coinjected with Px1 RNA together with equal amounts of RNAs encoding either Px2 (red traces) or the W77R mutation of human Cx26 (black traces), which is devoid of functional activity (31). (C and D) Coexpression of Px1 and Px2 reduced the amplitude of the outward currents induced by the depolarizing voltage steps (bottom traces). ΔI_m recorded from Px1/Px2 (red circles) expressing oocytes was significantly less (^*, P < 0.001) than that measured from Px1/W77R cells (black circles). Results are shown as mean ± SEM from four independent experiments. Antisense (n = 39); Px1/W77R (n = 60); Px1/Px2 (n = 67). (E) Px1/Px2 channels exhibit a delayed peak current time. Oocytes were depolarized to +40 mV (top left traces) and +60 mV (top right traces) from a holding potential of -40 mV. Peak currents were reached with a significant delay after the imposition of the voltage step (32 and 68 msec at +60 mV and 62 and 96 msec at +80 mV, for Px1/W77R and Px1/Px2, respectively). The lower panels show the mean ± SEM from three independent experiments for Px1/W77R (n = 45) and Px1/Px2 (n = 50); ^*, P < 0.001. (F) Px2 slows the kinetics of voltage-dependent closure of Px1 hemichannels. Cells were depolarized to +60 mV from a holding potential of -40 mV (Upper). Px1/Px2 hemichannels (red) gated more slowly than those formed by Px1/W77R (black). The time-dependent decline in I_m was well fit by a first-order exponential decay function (Left Lower; cyanide line superposed to the rescaled current traces shown above). (Right Lower) The mean ± SEM from three independent experiments, for Px1/W77R (n = 44) and Px1/Px2 (n = 41); ^*, P < 0.001.