Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Abstract

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

Fig. 1.

Alignment of acid-sensing ion channel 1 (ASIC1) isoforms from different species. Sequence alignment of ASIC1 isoforms from the human (hASIC1a, NM_020039; and hASIC1b, NM_001095), mouse (mASIC1a, NM_009597; and mASIC1b, AB208022), and rat (rASIC1a, NM_024154; and rASIC1b, AJ309926) was done with Clusta IW, and shading was done with the Boxshade program (http://www.ch.embnet.org/). Identical amino acids are shown as white letters on a black background, and conserved amino acids are shown as black letters on a shaded background. The positions of the transmembrane domains were obtained from Jasti et al. (26) and are shown by the black horizontal bars. The amino acid sequence of hASIC1b was analyzed with Genetics Computer Group (University of Alabama at Birmingham) and Scansite (MIT) software for consensus PKC phosphorylation sites. The three consensus PKC phosphorylation sites on hASIC1b are shown (★), and they are T26, S40 (on the cytoplasmic NH₂ terminus), and S499 (on the cytoplasmic COOH terminus).

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 measured by two-electrode voltage clamp (TEV) at 1–4 days postinjection as described in materials and methods. A: representative traces of acid-activated currents of WT hASIC1b and each hASIC1b mutant. B: peak pH 4.0 current (I_pH4.0) for each phosphorylation mutant was normalized to the average peak I_pH4.0 recorded on the same day in oocytes of the same batch expressing WT hASIC1b. Shown is the summary of 3–6 experiments; values are means + SD. The number of individual oocytes recorded is shown in parentheses on top of each bar. P values were determined with one-way ANOVA with Tukey's post-hoc test. *P < 0.01 and †P < 0.001 vs. WT. NC, no acid-activated current. C: pH activation curves for WT hASIC1b and hASIC1b phosphorylation mutants. Peak acid-activated currents were recorded by switching the extracellular solution to pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.0 sequentially, as described in materials and methods, and were normalized to peak I_pH4.0. Normalized values (I/I_pH4.0) were fitted to the Hill equation to obtain pH₅₀ values and Hill coefficients. n = 13 oocytes for WT, 10 oocytes for S40A, 11 oocytes for S499A, 8 oocytes for S40A/S499A, 8 oocytes for S40E, 8 oocytes for S499D, 5 oocytes for S40E/S499A, and 5 oocytes for S40E/S499D. T26A (n = 8) and T26E (n = 8) constructs did not exhibit any acid-activated current at any pH (data not shown).

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 μg) was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and immunoblotted with an antibody to ASIC1 (1:200) or to actin (1:5,000) as a loading control. The blot shown is representative of 2–4 experiments comparing WT hASIC1b expression with that of each hASIC1b phosphorylation mutant.

Fig. 4.

PKC activators PMA and phorbol 12,13-dibutyrate (PdBu) reduce acid-activated currents of WT hASIC1b 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 measured by TEV before and after the addition of 1 μM PMA or 1 μM PdBu to the bath for 5 min to activate endogenous oocyte PKC. The negative controls used were either no treatment, vehicle (DMSO, 1:1,000), or an inactive PMA analog (4-α-PMA, 1 μM) added to the bath for 5 min between current measurements. Bar graph values are fractions of peak I_pH4.0 recorded after treatment over peak I_pH4.0 recorded before the treatment. Representative acid-activated current traces before (solid line) and after (shaded line) 1 μM PMA was added to the bath are also shown to the right of each graph. The numbers in parentheses on top of each bar indicate the number of oocytes. P values were determined by a two-tailed paired Student's t-test comparing the sets of peak I_pH4.0 values before and after each corresponding treatment. Data are means + SD.

Fig. 5.

PMA inhibits peak acid-induced currents of hASIC1b in transfected Chinese hamster ovary (CHO)-K1 cells. A: in outside-out patches of CHO-K1 cells transfected with a bicistronic plasmid encoding WT hASIC1b and enhanced green fluorescent protein, acid-induced currents were observed. Treatment with 100 nM PMA significantly reduced the current. B: for analysis, currents were normalized to address expression variability and patch size. Data are shown as average normalized currents for 3 pulses before PMA application and 3 pulses 5 min post-PMA application; n = 3 cells. Data were analyzed with a paired Student's t-test.

Fig. 6.

The PKC inhibitor chelerythrine abolishes the effect of PMA on WT and S499A hASIC1b. A: oocytes expressing WT hASIC1b were pretreated with the PKC inhibitor chelerythrine (1 μM) for 1 h, and peak I_pH4.0 was measured before and after 5 min of 1 μM PMA, 1 μM 4-α-PMA, DMSO (1:1,000), or no treatment. Shown are representative pH 4.0-activated currents recorded by TEV before (solid line) and after the addition of 1 μM PMA to the bath for 5 min (shaded line) in oocytes expressing WT hASIC1b pretreated with 1 μM chelerythrine for 1 h. B: oocytes expressing S499A hASIC1b were pretreated with 1 μM chelerythrine overnight. Peak pH 4.0-activated currents were measured before and after the addition of 1 μM PMA to the bath. Peak I_pH4.0 recorded after 5 min of treatment was normalized to peak I_pH4.0 recorded before treatment on the same oocyte. The effect of PMA on control untreated oocytes is also shown. Bar graphs represent mean normalized values + SD. P values shown on top of each bar were determined with two-tailed paired Student's t-test on sets of before and after peak I_pH4.0 values for each treatment. The number of oocytes is shown in parentheses.

Fig. 7.

Effect of PKC inhibitors on acid-activated currents of WT, S40A, S499A, or S40A/S499A hASIC1b. Oocytes were injected with 11.5 ng RNA for WT hASIC1b, S40A, S499A, or S40A/S499A. Two to three days postinjection, they were pretreated with 1 μM chelerythrine for 1 h (A–D) or injected with PKC inhibitory peptide 19–31 (PKC IP 19–31) at a final concentration of 6 μM for 1 h (A). pH 4.0-activated hASIC1b currents were recorded by TEV. Bar graph values are average peak I_pH4.0 from chelerythrine-treated oocytes or oocytes injected with PKC IP 19–31 normalized to average peak I_pH4.0 from untreated oocytes or oocytes injected with vehicle for PKC IP 19–31 (5% acetic acid). Data are summaries of 3–6 experiments, and values are means of individual oocytes + SD. The number of oocytes is shown in parentheses. P values were determined using a two-tailed unpaired Student's t-test vs. control.

Fig. 8.

PMA and chelerythrine have no effect on the total expression or surface expression of hASIC1b. A and B: immunoblots showing hASIC1b expression with and without PMA (A) or chelerythrine (B) treatment in total membranes prepared as described in materials and methods 3 days postinjection of oocytes with RNA for WT, S40A, or S499A hASIC1b. U, uninjected oocytes. Equivalent amounts of protein (30 μg) were loaded on each lane and separated by SDS-PAGE. Proteins were transferred to PVDF membranes, which were blotted for hASIC1 (1:200) and actin (1:5,000) as a loading control. As shown by the actin immunoblot, these experiments were repeated 2–4 times. C: mean normalized luminescence of oocytes expressing hemagglutinin (HA)-tagged WT hASIC1b. There was no statistically significant difference between WT hASIC1b surface expression in control or treated oocytes (P = 0.628 by one-way ANOVA). Shown is a summary of 3 experiments. The number in parentheses on top of each bar indicates the number of individual oocytes measured.