Testing the involvement of PKC in the prolonged recovery from inactivation produced by PMA.

<p><b>(A)</b> Cells were pretreated with calphostin C (200 nM) or a mixture of bisindolylmaleimide-1 (Bis; 500 nM) + Go6983 (100 nM). Inactivation was produced by a stimulus train (5Hz for 10s as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134117#pone.0134117.g004" target="_blank">Fig 4</a>) and the fractional recovery from inactivation determined first in the absence and then in the presence of PMA. Solid lines show an exponential fit to the mean data (left panel: calphostin A = 0.95, t = 17.2 s; calphostin + PMA A = 1.0, t = 23.7 s, comparison of fits F = 3.62 p = 0.06: right panel: Bis/Go A = 0.80, t = 15.6 s; Bis/Go + PMA A = 0.67 t = 40.7 s, comparison of fits F = 25.3 p < 0.0001). The arrows labeled “R₄₀“denote the 40s recovery time point. <b>(B)</b> Plots the mean recovery time constant determined from an exponential fit in each cell before (left panel) and during (right panel) application of PMA, or the inactive control 4α-PMA. “Control” = no pretreatment (n = 6); “PKCi” = cells recorded with intracellular application of a pseudosubstrate peptide inhibitor of PKC (2 μM PKC(19–36) (n = 6); “Bis + Go” = cells pretreated with bisindolylmaleimide-1 (500nM) + Go6983 (100nM) (n = 6); “Calphos” = cells pretreated with calphostin C (200 nM) (n = 7). Also shown is data for cells treated with 4α-PMA (n = 4). Pretreatment with the various drugs did not significantly alter the recovery time constant before application of PMA (left panel). The effect of PMA was significantly reduced by pretreatment with Bis + Go or calphostin C (right panel) (<i>ns</i>, not significantly different, * p < 0.05, *** P < 0.001 compared to the control PMA cells (red bar) determined using one-way ANOVA and Dunnett’s post-test). (<b>C</b>) To quantify the change in recovery rate produced by PMA, a ratio of the recovery time constants was calculated in each cell (tau in the presence of PMA / tau before application of PMA). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, ** P < 0.01). <b>(D)</b> Another index to compare the various drug treatments is the percent inhibition of recovery at the 40 s time point (R₄₀—see panel A and results section for more detail). Statistical significance compared to the control PMA cells (red bar) was determined using one-way ANOVA and Dunnett’s post-test (ns, not significantly different, * p < 0.05, *** P < 0.001).