Fig. 4. mDia and HDAC6 mediate α1D-AR activation of PANX1.

a, b Basal and PE-stimulated whole-cell currents in α1D-AR/hPANX1(TEV)-transfected HEK293T cells co-expressing a wild-type diaphanous autoregulatory domain (DAD) construct (a) or a mutated DAD(M1041A) version that cannot bind mDia (b). c Summary data (mean ± s.e.m) reveal that DAD, but not DAD(M1041A), increased basal PANX1 current and occluded PE effects. n = 11, 10, or 7 cells examined over 5 independent experiments. Two-way ANOVA (F_2,25 = 5.673, p = 0.0093) with Holm-Sidak’s multiple comparisons test (p values from comparisons are shown). n.s. not significant. d, e Basal and PE-stimulated whole-cell currents in α1D-AR/hPANX1-transfected cells recorded with vehicle (DMSO, 14.1 µM) or tubacin (2 μM) in the pipette. f Concentration-dependent inhibition of PE-induced hPANX1 current by tubacin. n = 7 cells per group examined over 5 independent experiments. Data are presented as mean ± s.e.m. Two-way ANOVA (F_2,18 = 7.636, p = 0.0040) with Bonferroni’s multiple comparisons test. g, h Effects of tubacin (2 µM) treatment on basal whole-cell currents (mean ± s.e.m in α1D-AR/hPANX1(TEV)-transfected cells co-expressing the mDia-activating constructs, RhoA(G14V/F39A) (g) or DAD (h). Tubacin was added in culture media after transfection, and to the intracellular solution for whole-cell recording. Insets: representative time series of whole-cell current at 80 mV from α1D-AR/hPANX1(TEV)-transfected cells, with RhoA(G14V/F39A) or DAD, treated with tubacin. i Summary data (mean ± s.e.m) reveal that PE-induced CBX-sensitive currents from α1D-AR/hPANX1(TEV)-expressing cells were diminished by co-expression of RhoA(G14V/F39A) or DAD, regardless of tubacin treatment. n = 11, 9, 8, or 10 cells examined over 6 independent experiments. Dotted line and shaded area indicate mean and SEM of PE-evoked, CBX-sensitive current density from cells expressing α1D-AR and hPANX1. PE phenylephrine, CBX carbenoxolone.