0 [0.43 (SD 0.31), n = 38, P < 0.001, 95% CI (0.327, 0.532), for S499A and 0.58 (SD 0.15), n = 14, P < 0.01, 95% CI (0.498, 0.670), for S499D]. The double mutant construct S40A/S499A exhibited FTY720 IC50 reduced functional expression as well [0.39 (SD 0.46), n = 19, P < 0.001, 95% CI (0.170, 0.161)]. The S40E/S499A mutation had an even greater effect [0.0877 (SD 0.137), n = 17, P < 0.001, 95% CI (0.0172, 0.158), P < 0.05 vs. S499D]. The S40E/S499D mutation also expressed reduced IpH4.0 compared with WT [0.330 (SD 0.271), n = 17, P < 0.001, 95% CI (0.191, 0.470)]. We were not able to measure any acid-activated currents in oocytes expressing T26A (n = 8) or T26E (n = 11) hASIC1b constructs. The translation of these constructs in oocytes was confirmed by immunoblot analysis of total membranes of uninjected oocytes or oocytes injected with each of the ASIC1b constructs with an ASIC1 antibody (Fig.

3). Fig. 2. Mutations in PKC consensus phosphorylation sites on hASIC1b affect its function in Xenopus oocytes. cRNA (11.5 ng) for wild type (WT) hASIC1b or hASIC1b PKC phosphorylation mutants was expressed in Xenopus oocytes. Acid-activated currents at pH 4.0 were … Fig. 3. hASIC1b expression in Xenopus oocytes. Oocytes were injected with 11.5 ng RNA for WT hASIC1b or each hASIC1b mutant, and total membranes were isolated as described in materials and methods. Protein (30 ��g) was resolved by SDS-PAGE, transferred … Because ASIC1 is a ligand-gated channel, activated by H+, we determined if the mutations in the consensus PKC phosphorylation sites have any effect on the affinity of the channel for protons.

Oocytes were injected with cRNA for each hASIC1b mutant, and acid-activated currents were recorded at different activation pHs ranging from pH 7.0 to 4.0 (Fig. 2C). The oocyte was sequentially exposed to solutions of higher pH and then to lower pH. To determine if hASIC1b current exhibited rundown, i.e., a diminution of the peak current with repeated acid challenges, two consecutive activation curves were taken, and no significant rundown of the current was observed (not shown). Peak acid-activated currents at each pH were normalized to peak IpH4.0 (Fig. 2C). pH50 values of activation and Hill coefficients were determined for each individual oocyte by fitting normalized current (I)/IpH4.0 at each test pH to the sigmoidal dose-response (variable rate) equation in Prism 3. The pH activation curve shown is the average of all the oocytes. Means and Dacomitinib SDs of pH50 values and Hill coefficients are shown in Table 1. pH50 values of hASIC1b phosphorylation mutants were not significantly different than the pH50 value of WT hASIC1b. However, there was a statistically significant difference in Hill coefficients (Table 1). Table 1.