Figure 6: Single-channel activity of PANX1 is gradually increased during apoptosis and upon α1D adrenoceptor-mediated activation.

(a) Cell-attached recording from anti-Fas-treated apoptotic Jurkat T cells after inhibition of caspase activity at 60 min using 25 μM Q-VD-OPh (Q-VD); two CBX-sensitive channels with unitary conductance of either ∼82 pS (O_L1) or ∼58 pS (O_S1) were present in this patch. (b) Unitary conductance for CBX-sensitive PANX1 channels in apoptotic Jurkat T cells, obtained from cells treated with Q-VD at 30, 60 or 120 min of anti-Fas exposure. Data obtained from the same patch are labelled in the same colour. Shaded region represents 95% confidence interval of unitary conductance obtained from C-terminally truncated PANX1 in cell-attached configuration (from d; Supplementary Fig. 1d). (c) Cell-attached recording from HEK293T cells co-expressing α1D adrenoceptors (α1DR) and full-length PANX1-GFP channels obtained at +80 and +60 mV. PANX1 channel activity was not observed before phenylephrine (PE, 20 μM); small conductance channel openings (O_S1, ∼23 pS) observed after ∼4 min of PE exposure showed lower open probability after CBX (see boxed region). A larger conductance channel (O_L1, ∼80 pS) became predominant in the patch after ∼8 min exposure to PE (blue arrows). (d) Outwardly rectifying steady-state single-channel I–V relationships (mean±s.e.m.) from cell-attached recordings of C-terminally truncated PANX1 (red) or α1DR-activated PANX1 (blue), with averaged unitary conductance of 74.7±3.6 pS or 82.7±1.4 pS at positive potentials (n=8 and 4), and 12.8±1.2 pS and 14.9±2.1 pS at negative potentials (n=4 and 3). (e,f) Representative cell-attached recordings at +80 mV from HEK293T cell expressing GFP-tagged, C-terminally truncated PANX1 (e) or α1DR and full-length PANX1 (f), ∼12 min after PE application. (g) Open time distribution from the cell-attached recordings depicted in e,f; mean open time for C-terminally truncated and α1DR-activated PANX1-GFP are ∼7.9 and ∼1.3 ms.