We examined the effect of PKC activation with the phorbol esters PMA or PdBu on selleckchem hASIC1b currents. PMA and PdBu are cell-permeable diacylglycerol (DAG) analogs that activate classical and novel PKC isoforms. Oocytes were injected with WT hASIC1b, S40A hASIC1b, or S499A hASIC1b to examine the importance of the amino acids that are consensus PKC phosphorylation sites. Acid-activated currents were recorded in the same oocyte before and after 1 ��M PMA or 1 ��M PdBu addition to the bath solution for 5 min. Incubation of the oocyte with PMA decreased IpH4.0 of WT hASIC1b [0.70 (SD 0.20), n = 29, P = 0.0008 by two-tailed paired t-test of IpH4.0 before and after treatment]. PdBu also inhibited WT hASIC1b current [0.67 (SD 0.12), n = 6, P = 0.010 by two-tailed paired t-test].

Incubation of WT hASIC1b-injected oocytes in the bath solution (ND96 pH 7.4 solution) for 5 min without treatment (n = 27), with DMSO (1:1,000, n = 10), or with the inactive PMA analog 4-��-PMA (1 ��M, n = 12) had no effect on acid-activated currents (Fig. 4A). The 95% CIs for the means of the ratio of IpH4.0 after treatment to IpH4.0 before treatment for the WT hASIC1b construct were (0.940, 1.09) for 5 min of no treatment, (0.852, 1.05) for DMSO, (0.856, 1.19) for 4-��-PMA, (0.626, 0.783) for PMA, and (0.544, 0.792) for PdBu. A statistically significant effect of PMA or PdBu on hASIC1b currents was not observed in oocytes expressing S40A hASIC1b [0.87 (SD 0.13), n = 8, P = 0.061 by two-tailed paired t-test for PMA treatment and 0.80 (SD 0.059), n = 4, P = 0.10 for PdBu treatment; Fig. 4B].

The 95% CIs for the ratios of IpH4.0 after treatment to IpH4.0 before treatment for the S40A construct were (0.799, 1.05) for 5 min of no treatment, (0.768, 0.984) for DMSO, (0.922, 1.00) for 4-��-PMA, (0.766, 0.978) for PMA, and (0.7, 0.89) for PdBu. The S499A hASIC1b construct responded to the activation of endogenous oocyte PKC with PMA or PdBu in a similar manner to WT hASIC1b, decreasing by a similar amount [0.64 (SD 0.24), n = 8, P = 0.033 by two-tailed paired t-test for the PMA effect and 0.55 (SD 0.23), n = 3, P = 0.064 by two-tailed paired t-test for the PdBu effect; Fig. 4C]. The 95% CIs for the mean ratios of IpH4.0 after treatment to IpH4.0 before treatment for the S499A channel were (0.914, 1.09) for 5 min of no treatment, (0.645, 1.15) for DMSO, (0.844, 1.01) for 4-��-PMA, and (0.

461, 0.824) for PMA. Fig. 4. PKC activators PMA and phorbol 12,13-dibutyrate (PdBu) reduce acid-activated currents of WT hASIC1b Cilengitide and S499A hASIC1b but not that of S40A hASIC1b. Oocytes were injected with RNA for WT hASIC1b (A), S40A (B), or S499A (C). Acid-activated currents were … Because PKC can have cell type-specific effects (11), we repeated the experiment of activating PKC with PMA in CHO cells transfected with WT hASIC1b-eGFP bicistronic vector. Acid-activated currents were recorded before and after the superfusion of 100 nM PMA in the chamber.