Signaling Pathways in Proton and Non-proton ASIC1a Activation

Abstract

Acid-sensing ion channels (ASICs) regulate synaptic activities and play important roles in neurodegenerative diseases as well as pain conditions. Classically, ASICs are described as transiently activated by a reduced pH, followed by desensitization; the activation allows sodium influx, and in the case of ASIC1a-composed channels, also calcium to some degree. Several factors are emerging and extensively analyzed as modulators, activating, inhibiting, and potentiating specific channel subunits. However, the signaling pathways triggered by channel activation are only starting to be revealed.The channel has been recently shown to be activated through a mechanism other than proton-mediated. Indeed, the large extracellular loop of these channels opens the possibility that other non-proton ligands might exist. One such molecule discovered was a toxin present in the Texas coral snake venom. The finding was associated with the activation of the channel at neutral pH via the toxin and causing intense and unremitting pain.By using different pharmacological tools, we analyzed the downstream signaling pathway triggered either by the proton and non-proton activation for human, mouse, and rat ASIC1a-composed channels in in vitro models. We show that for all species analyzed, the non-protonic mode of activation determines the activation of the ERK signaling cascade at a higher level and duration compared to the proton mode.This study adds to the growing evidence of the important role ASIC1a channels play in different physiological and pathological conditions and also hints at a possible pathological mechanism for a sustained effect.

Keywords: ASIC1a; ERK; MitTx; non-proton activation; pain; proton activation.

Figure 1

Non-proton activation of ASIC1a in striatal neurons. Representative membranes of lysates from 7 DIV wild type (A), and ASIC1a knockout (B) C57 mice striatal neuronal cultures were incubated with MitTx and compared to treatment with pH6 solutions for the time indicated (2, 10, or 30 min). The detection was performed with anti ASIC1, tubulin (Tub), phospho ERK (pERK), total ERK (tERK), or phospho CaMKII (pCaMKII) antibodies, and Licor secondary antibodies. (C) Examples of images of striatal cultures used and treated with MitTx and stained with tubulin (red) and phosphoERK (green) antibodies and secondary Alexa fluor antibodies, 60× objective used. Scale bar 10 μm. (D) Result of the bands detected in (A) for pERK/ERKt levels relative to control samples showing an increase in both, pH6 or MitTx treatments and the same pattern of increase for pCaMKII. Notice the lack of effect in ASIC1a knock-out derived cultures. (A) Notice that plots are the result of the signal intensity of the bands detected for each antibody, and tERK and tubulin are used as loading controls between loaded samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, ****p < 0.0001; ***p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control (Ctr) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 3.77 ± 0.07; pH6 30’ 1.32 ± 0.09; MitTx 2’ 5.39 ± 0.08. For pCaMKII/Tub: pH6 2’ 4.97 ± 0.14; pH6 30’ 1.20 ± 0.07; MitTx 2’ 6.37 ± 0.28.

Figure 2

Proton and non-proton activation of ASIC1a in HEK cells. (A) Representative membranes of lysates of HEK cells treated with pH6 or MitTx for 2 or 10 min or preincubated with Pctx-1 compared to untreated cells (control, Ctr) and detected with phosphoERK (pERK), total ERK (tERK), and ASIC1 antibodies. (B) Representative membrane of the same lysates to detect pCaMKII levels. (C) Plots showing detected levels of pERK (top panel) or pCaMKII (lower panel). Notice that the increase in kinase levels goes further at a later time point (2 vs. 10 min) in MitTx treated cultures compared to pH6 treated ones that show an increase at 2 min followed by a reversal to control levels consistent with the proton-activated desensitizing mechanism. (A) Notice that plots are the result of the signal intensity of the bands—with tERK and tubulin used as loading controls between loaded samples—and expressed relative to control samples. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments against the control were performed, mean values above bars; n = 3 membranes, ****p < 0.0001; ***p 0.0001–0.001; ns: no significant differences. Mean values expressed relative to control (Ctr) levels ± SEM are as follows: for pERK/tERK: pH6 2’ 6.10 ± 0.13; pH6 10’ 1.23 ± 0.11; pH6 2’ Pctx 1.39 ± 0.08; MitTx 2’ 7.98 ± 0.10; Mittx10’ 10.90 ± 0.17; MitTx 2’ Pctx 1.18 ± 0.05; MitTx 10’ Pctx 1.42 ± 0.05. For pCaMKII/Tub: pH6 2’ 7.31 ± 0.21; pH6 10’ 1.16 ± 0.06; pH6 2’ Pctx1.14±0.07; Mittx 2’ 8.88 ± 0.15; Mittx 10’ 10.76 ± 0.32; Mittx 2’ Pctx 1.17 ± 0.07.

Figure 3

Proton and non-proton activation of overexpressed ASIC1a channels. (A) Representative membranes of lysates of cells control (ctr) or transfected with eGFP-ASIC1a (eASIC) at two levels (1x or x3) to obtained different levels of expression of the protein (“eASICx1 or eASICx3”), and treated with pH6 or MitTx with or without pre-incubation of Pctx-1 or untreated. (B) Plots showing the increase in pERK and pCaMKII levels calculated from membranes as that shown in (A), consistent with the increase in eASIC expressed. Notice the level of increase achievable via MitTx incubation at the highest overexpressed level of eASIC, higher than that obtained via pH6. (C) Representative membrane showing the different levels of eASIC in cells overexpressing the channel (1x or 3x), detected with an ASIC1 antibody. (D) Comparison between the different ASIC1 proteins expressed (the endogenous human ASIC1a; of approx. 67 kDa) and the overexpressed eASIC (approx. 110 kDa, and expressed at different levels; x1 or x3). (A) Notice that plots are the result of the signal intensity of the band detected,—tERK and tubulin are used as loading controls between loaded samples—and expressed relative to eASICx1 levels. Data are presented as the mean ± SEM ANOVA and Dunnet post hoc test for treatments and conditions were performed, mean values above bars; n = 3 membranes, ****p < 0.0001; ns: no significant differences. Mean values expressed relative to eASICx1 levels ± SEM are as follows: eASICx3 3.16 ± 0.06; eASIC MitTx 3.42 ± 0.15; eASICx3 MitTx 9.96 ± 0.12; eASIC MitTx Pctx 1.10 ± 0.05; eASIC pH6 2’ 1.85 ± 0.08; eASIC pH6 2’ Pctx 1.08 ± 0.05; eASICx3 pH6 2’ 4.00 ± 0.12.