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. Author manuscript; available in PMC: 2013 Dec 24.
Published in final edited form as: Cell Rep. 2013 Nov 14;5(4):10.1016/j.celrep.2013.10.015. doi: 10.1016/j.celrep.2013.10.015

Fig. 4. δOR and ORL1 inhibition of VDCCs is regulated by LIMK, ROCK and actin polymerization.

Fig. 4

δOR: A. The δOR agonist, SNC80, activates cofilin: Wild-type MEFs (wt MEFs) or β-arr1−/− MEFs were treated with SNC80 for 0–120min and lysates analyzed by Western blot. ai. Representative western blots of phosphorylated cofilin (pcof) and total cofilin (tcof) in wild type wt MEFs (upper two panels) and βarr1−/− MEFs (lower two panels) after SNC80 treatment. aii. SNC80 (1μM) decreased phospho-cofilin after 2, 5, 15 and 30min in wt MEFs (closed circles) whereas βarr1−/− MEFs (open circles) showed no change. Values on the y-axis represent phospho-cofilin normalized to basal levels at each time point shown on the x-axis. a; p<0.001, b: p<0.01, c: p<0.01 vs untreated wt MEFs. B i. The ROCK inhibitor, Y27632 (200 and 400ng/ml) reduced δOR-VDCC coupling in β-arr1−/− and +/+ neurons. Excess LIMK (2μg/ml) increased SNC80-VDCC inhibition in both β-arr1 +/+ and −/− neurons although SNC80-VDCC inhibition remained higher in β-arr1−/− neurons. LIMK phosphorylation of cofilin can be prevented by the Serine 3 peptide. This decreased SNC80-VDCC inhibition in β-arr1−/− neurons but not +/+ neurons (S3: 1.6 and 6.4ng/ml). C. i. Jasplakinolde and Thymosin B4 (200ng/ml), enhancers of actin polymerization, increased SNC80-VDCC inhibition in β-arr1+/+ to −/− levels whereas Latrunculin B (LatB: 200ng/ml), an inhibitor of actin polymerization, reversed the increased δOR-VDCC inhibition in β-arr1−/− to +/+ levels. ii. LatB did not alter GABAB-VDCC inhibition assessed by Baclofen (50μM). ORL1: D. The increase in ORL1-VDCC inhibition in β-arr1−/− DRG neurons was reversed by the ROCK inhibitor, Y27632 (400ng/ml), the LIMK inhibitory peptide, S3 (6.4ng/ml), and Latrunculin B (200ng/ml). No effect of these manipulations was seen in +/+ neurons. All data are shown as mean±SEM, *p<0.05, ***p<0.001 vs β-arr1+/+, ##, ###p<0.01, or 0.001 vs untreated, matched genotype, n=6–12 per data point. All exemplar currents depict the current before, during and after agonist application (1, 2 and 3) and the scale bars show time, 20ms, and current, 5nA on the x- and y-axes respectively. Columns in dark (β-arr1+/+) or light (β-arr1−/−) shades of grey indicate data shown in Fig 1.