4 solution for 10 min. Each oocyte was then placed in a 1.5-ml Eppendorf tube, and all the solution was removed. ELISA Supersignal West nearly Femto Chemiluminescence substrate (50 ��l, Pierce) was added, and luminescence was measured with a Turner TD20/20 luminometer (Turner BioSystems, Sunnyvale, CA). Data analysis and statistics. Data are presented as means (SD). Statistical significance was determined with two-tailed paired or unpaired Student’s t-tests (in Excel) for two-group comparisons or one-way ANOVA followed by Tukey’s post hoc test for comparisons of three or more groups (GraphPad Prism 3). A statistically significant difference was accepted if P < 0.05; 95% confidence intervals (CIs) were also determined for each mean (GraphPad Prism 3). RESULTS Mutations in hASIC1b consensus PKC phosphorylation sites and hASIC1b expression.
We analyzed the amino acid sequence of hASIC1b for PKC phosphorylation motifs with GCG (UAB) and Scansite (MIT) software. This analysis resulted in several consensus PKC phosphorylation sites; however, we decided to concentrate on the three amino acids that are located on the intracellular NH2 and COOH termini of hASIC1b: T26, S40, and S499 (Fig. 1) because the phosphorylation of membrane proteins is an intracellular event (43). We used site-directed mutagenesis to generate phosphorylation mutants at each of these three sites. Mutations to alanine prevent phosphorylation, and mutations to glutamic acid or aspartic acid can often mimic a phosphorylated amino acid. We first assessed the effect of these phosphorylation site mutations on the functional expression of each construct in Xenopus oocytes.
Oocytes were injected with WT hASIC1b or mutant cRNA for each phosphorylation site. Electrophysiological recordings were obtained 1�C4 days postinjection. Oocytes were voltage clamped at ?60 mV, and acid-activated currents were recorded by rapidly switching the solution bathing the oocyte from ND96 pH 7.4 to ND96 pH 4.0 solution. Representative traces of these recordings are shown in Fig. 2A. Uninjected oocytes did not exhibit any acid-activated currents (not shown; also, there is no acid-activated current in oocytes injected with two of the hASIC1b mutants in Fig. 2). Peak IpH4.0 of each mutant was normalized to the average peak IpH4.0 of WT hASIC1b constructs recorded from the same batch of oocytes on the same day postinjection [1.
01 (SD 0.39), n = 85, 95% CI (0.925, 1.10); Fig. 2B]. Mutation of S40 to alanine had no effect on the functional expression of hASIC1b, as S40A exhibited average peak pH 4.0-activated currents that were not statistically different than WT hASIC1b [0.88 (SD 0.63), n = 24, P = not significant (NS) by one-way ANOVA, 95% CI (0.609, 1.15), P < 0.05 vs. S499D]. Mutation of S40 to glutamic acid, a phosphorylation mimic, resulted in a decrease in IpH4.0 of hASIC1b [0.39 (SD 0.42), n = 28, P < 0.001, GSK-3 95% CI (0.231, 0.552)].
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