Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions - PubMed (original) (raw)
Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions
Anne Delaunay et al. Proc Natl Acad Sci U S A. 2012.
Abstract
In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particularly important sensor of nonadaptive pain associated with tissue acidosis. However, little is known about the human ASIC3 channel, which includes three splice variants differing in their C-terminal domain (hASIC3a, hASIC3b, and hASIC3c). hASIC3a transcripts represent the main mRNAs expressed in both peripheral and central neuronal tissues (dorsal root ganglia [DRG], spinal cord, and brain), where a small proportion of hASIC3c transcripts is also detected. We show that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to directly sense extracellular pH changes not only during acidification (up to pH 5.0), but also during alkalization (up to pH 8.0), an original and inducible property yet unknown. When the external pH decreases, hASIC3 display a transient acid mode with brief activation that is relevant to the classical ASIC currents, as previously described. On the other hand, an external pH increase activates a sustained alkaline mode leading to a constitutive activity at resting pH. Both modes are inhibited by the APETx2 toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of hASIC3 is an intrinsic property of the channel, which is supported by the extracellular loop and involves two arginines (R68 and R83) only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically adapt its activity between pH 5.0 and 8.0, a property likely to participate in the fine tuning of neuronal membrane potential and to neuron sensitization in various pH environments.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Relative expression of ASIC mRNAs in human neuronal tissues. Quantitative RT-PCR experiments performed on total RNA from human DRG, brain, and spinal cord. (Inset) relative expression of ASIC3 in rat DRG and brain as a comparison. Expression is normalized to the expression of ASIC3 in DRG.
Fig. 2.
Macroscopic properties of the human ASIC3 currents. Whole-cell recordings of hASIC3a currents performed at −80 mV from F-11 transfected cells. Currents were activated by rapid changes of the external pH from pH 8.0 to 7.0, as indicated above each current trace. Dashed lines represent the zero current level. The nonconventional current (main trace) and the classic current (Inset) are represented.
Fig. 3.
Properties of the nonconventional hASIC3a sustained current in F11 cells. (A) Amplitudes of the hASIC3a sustained current component (Isst) for the classic (white bars) and nonconventional (black bars) currents recorded at different external pH (HP -80 mV). The amplitudes were measured as the difference between the current level reached at the test pH (7.7, 7.4, 7.0, 6.6, and 5.0) and the current level at the holding pH 8.0 (see Inset). The number of experiment is indicated above each bar (**P < 0.01 and ***P < 0.001, Mann–Whitney test). (B) Comparison of the amplitude of the basal current measured at the holding pH 8.0 in cells expressing the classic and the nonconventional hASIC3a currents (HP -80 mV, ***P < 0.001, Kruskal–Wallis test followed by a Dunn’s post hoc test). As illustrated in the Inset, the APETx2 toxin at 1 μM inhibited the basal current in cells expressing the nonconventional hASIC3a current. (C) The macroscopic membrane conductances G, calculated from 10-mV pulses applied to cells expressing the nonconventional hASIC3a current (Inset), are represented as a function of external pH both in control conditions (black bars) and in the presence of the APETx2 toxin at 1 μM (gray bar; *P < 0.05, **P < 0.01, and ***P < 0.001, repeated measures ANOVA test). (D) Sensitivity of the pH 8.0–induced hASIC3a current to extracellular Na+ ions and APETx2 toxin (3 μM). The 0-Na condition was obtained by replacing external Na+ by _N_-Methyl
d
-Glucamine (NMDG+) ions. A voltage ramp protocol was applied to the current levels obtained in the presence (1) and in the absence (2) of APETx2 (3 μM). E, I/V curve of the pH 8.0–induced APETx2-sensitive hASIC3a current.
Fig. 4.
The alkaline-induced sustained current is independent of the expression system. (A) pH 6.6–evoked current recorded at −80 mV from COS cells transfected with hASIC3a. The dotted line represents the zero current level, and the typical alkaline-induced sustained current is magnified. (B) Xenopus oocytes injected with hASIC3a also display a typical alkaline-induced sustained current. The two current traces were recorded at −80 mV from the same oocyte. (C) pH 7.0–evoked current recorded at −80 mV from a mouse cortical neuron transfected with hASIC3a. The current displays typical alkaline-induced sustained activity. (D) pH-dependent activation of the hASIC3a nonconventional sustained currents recorded in F-11 cells (data from five different cells). Amplitudes of the sustained currents are normalized to the current measured at pH 8.0 (I/IpH8.0). (E) Typical effects of extracellular alkalization to pH 8.0 (from the physiological pH 7.4) on whole-cell current recorded at −80 mV from F-11 cells expressing either hASIC3a, rASIC3, or hASIC1a channels. The alkaline-induced current was not observed in rASIC3- or hASIC1a-expressing cells. rASIC3 showed a small widow current at pH 7.4 that was absent at pH 8.0.
Fig. 5.
Mapping of the structural elements involved in the alkaline sensitivity of hASIC3. (A) Effect of external alkalization (from pH 6.6 to 8.0) on oocytes expressing the rat ASIC3 chimera containing the extracellular loop of hASIC3a; (Upper) rASIC3-hLoop3 or the human ASIC3a chimera containing the extracellular loop of rASIC3; (Lower) hASIC3-rLoop3. (B) Schematic representation of two ASIC subunits in a functional channel. The two arginines only present in human ASIC3, and not found in rat, are indicated. Adapted by permission from ref. (Copyright 2007, Macmillan Publishers Ltd). (C) Effect of external alkalization on hASIC3a wild type and mutants. (D) Bar graph representing the effects observe in (C) (*P < 0.05 and ***P < 0.001, one-way ANOVA followed by Tukey’s post hoc test). (E) Bar graph of the amplitudes of the pH 5.0–induced transient currents measured from oocytes expressing the wild-type or the arginine mutants described in C and D.
Fig. 6.
Modulation of the hASIC3a alkaline-sensitive current. Whole-cell patch-clamp experiments performed on transfected F-11 cells clamped at −80 mV. (A) Effect of different external calcium concentrations on the basal sustained current recorded at pH8.0 in cells transfected with hASIC3a. The concentration of 1 μM free calcium was obtained by combining 5 mM EGTA and 4.97 mM CaCl2 at pH 8.0, as calculated with the maxchelator software (*P < 0.05 and **P < 0.01, Friedman test followed by a Dunn’s post hoc test). (B) Effect of lactate (20 mM) on the pH 8.0–induced hASIC3a sustained current. (C) Bar graph showing the potentiating effect of AA (10–20 μM) on the basal hASIC3a current recorded at pH 8.0 in cells displaying the alkaline sensitivity (Left; ***P < 0.001, Wilcoxon test). For comparison, the effect of AA of the basal current recorded at pH 8.0 from cells expressing rASIC3 is also represented (Right). (D) External application of AA (20 μM) was sufficient to trigger a hASIC3a current at physiological pH 7.4 in cells displaying the alkaline sensitivity. This AA-induced current was inhibited by APETx2 (5 μM, Inset). (E) Representative current trace showing the effect of arachidonic acid (10 μM) on a cell that initially displayed no pH 8.0–induced sustained current.
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