Human ASIC3 Channel Dynamically Adapts Its Activity to Sense the Extracellular Ph in Both Acidic and Alkaline Directions

Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions

Anne Delaunaya,b,c,1, Xavier Gasulla,d,e,1, Miguel Salinasa,b,c, Jacques Noëla,b,c, Valérie Frienda,b,c, Eric Linguegliaa,b,c,2,3, and Emmanuel Devala,b,c,2,3

aInstitut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientiﬁque, UMR 7275, 06560 Valbonne, France; bUniversité de Nice-Sophia Antipolis, 06560 Valbonne, France; cLabEx Ion Channel Science and Therapeutics, 06560 Valbonne, France; dNeurophysiology Laboratory, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036 Barcelona, Spain; and eInstitut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Rosello 149-153, 08036 Barcelona, Spain

Edited by Michael J. Welsh, Howard Hughes Medical Institute, Iowa City, IA, and approved June 28, 2012 (received for review December 12, 2011) In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has cutaneous acidification. Among ASICs, ASIC3-containing chan- recently emerged as a particularly important sensor of nonadap- nels are particularly interesting candidates for sensing the non- tive pain associated with tissue acidosis. However, little is known adaptive pain caused by protons. ASIC3 channels have the about the human ASIC3 channel, which includes three splice property to generate a sustained depolarizing current in response variants differing in their C-terminal domain (hASIC3a, hASIC3b, to moderate acidifications, and are able to integrate different in- and hASIC3c). hASIC3a transcripts represent the main mRNAs flammatory or ischemic stimuli such as protons, ATP, lactic and expressed in both peripheral and central neuronal tissues (dorsal arachidonic acid, and hypertonicity (16–19). All these properties root ganglia [DRG], spinal cord, and brain), where a small have been proposed to be important for the role of ASIC3 in pain proportion of hASIC3c transcripts is also detected. We show (16, 20, 21). Peripheral ASIC3-containing channels have been that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to shown (i) to participate to acidic, inflammatory, and postoperative fi directly sense extracellular pH changes not only during acidi ca- pain (16, 22, 23); (ii) to contribute to primary and/or secondary tion (up to pH 5.0), but also during alkalization (up to pH 8.0), an mechanical hyperalgesia in muscles and joints after inflammation original and inducible property yet unknown. When the external or injury (3, 24, 25); (iii) to be involved in cutaneous and visceral pH decreases, hASIC3 display a transient acid mode with brief mechano-sensation and mechano-nociception (24, 26, 27, 28); and activation that is relevant to the classical ASIC currents, as (iv) to support acid sensing in gastroesophageal afferents (29). previously described. On the other hand, an external pH increase Most of the studies on ASIC3 were performed in rodents and activates a sustained alkaline mode leading to a constitutive used rodent cDNAs. The detailed biophysical properties and activity at resting pH. Both modes are inhibited by the APETx2 tissue distribution of human ASIC3 remain poorly characterized. toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of We report here that human ASIC3 (hASIC3) not only sense hASIC3 is an intrinsic property of the channel, which is supported extracellular acidification but also extracellular alkalization. This by the extracellular loop and involves two arginines (R68 and R83) intrinsic capacity to behave as an acido-basic sensor brings, to- only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically gether with its wide distribution within the human nervous sys- adapt its activity between pH 5.0 and 8.0, a property likely to tem, an additional dimension to the role of hASIC3 channel in participate in the fine tuning of neuronal membrane potential and pain and other neurophysiological processes in humans. to neuron sensitization in various pH environments. Results

sodium channels | nociception Distribution of Human ASIC3 in Neuronal Tissues. In the rodent nervous system, ASIC3 is mostly expressed in sensory neurons (7), and functional studies have shown that native ASIC currents cid-sensing ion channels (ASICs) are depolarizing cationic – in central neurons are carried by ASIC1a and ASIC2 channels Achannels gated by extracellular protons (1 3). Four genes (30, 31). We performed quantitative RT-PCR experiments on encoding at least six subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, fi different human neuronal tissues to assess the relative abundance ASIC3, and ASIC4) have been identi ed so far in rodents. Func- of messenger RNAs for the three human isoforms hASIC3a, tional channels have been proposed to result from the trimeric hASIC3b, and hASIC3c. We found that the hASIC3a mRNA is association of subunits (4), leading to homomeric or heteromeric the main ASIC3 isoform expressed in human neuronal tissues, channels. ASICs are largely expressed in neurons, both in central although hASIC3c is also significantly detected (Fig. 1, see also and peripheral nervous systems. Whereas ASIC1a and ASIC2 are Fig. S1). The hASIC3b mRNA expression appears negligible in widely present in the rodent nervous system, the expression of all tested tissues. We therefore focused on hASIC3a in this study. ASIC1b and ASIC3 is primarily restricted to sensory neurons (5– 7). The ASIC3 subunit is highly expressed in rat nociceptive neurons (8, 9). The expression pattern of ASIC subunits is less well – Author contributions: X.G., M.S., E.L., and E.D. designed research; A.D., X.G., M.S., J.N., documented in humans, where ASIC3 (10 12) has three variants V.F., and E.D. performed research; A.D., X.G., M.S., J.N., V.F., E.L., and E.D. analyzed data; differing in their C-termini (2). The physiological relevance and and X.G., J.N., E.L., and E.D. wrote the paper. properties of these human variants have so far never been studied. The authors declare no conflict of interest. Several physiological and/or physiopathological conditions, This article is a PNAS Direct Submission. such as synaptic transmission, bone resorption, ischemia, in- 1A.D. and X.G. contributed equally to this work. flammation, tumor development, or tissue incisions, are accom- 2 fi E.L. and E.D. contributed equally to this work. panied by extracellular acidi cations. Moreover, tissue acidosis is 3To whom correspondence may be addressed. E-mail: [email protected] or lingueglia@ well known to be painful (13) and inhibition of ASICs in healthy ipmc.cnrs.fr. human volunteers (14, 15) has revealed the important role of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. these channels in sensing acid-induced pain provoked by 1073/pnas.1120350109/-/DCSupplemental.

13124–13129 | PNAS | August 7, 2012 | vol. 109 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1120350109 Downloaded by guest on September 30, 2021 7 unpaired t test). As previously described (11), the classic hASIC3a rASIC3 hASIC3a,b,c 1.0 hASIC3b current (Fig. 2, Inset) displayed a smaller plateau than its rat hASIC3c equivalent (Fig. S2A), because of a shift of the pH-dependent in- 6 0.5 hASIC1 activation of hASIC3a toward more alkaline values, which led to 4 hASIC2a a smaller window current. The transient phase of the classic and the expression hASIC2b

mRNA relave 0 DRG brain nonconventional hASIC3 currents displayed the same activation curve in response to extracellular acidiﬁcation (pH =6.56and 3 1/2

ve expression ve 6.59 with Hill slopes of 3.0 and 2.8, respectively; see Fig. S2B), and were both sensitive to the ASIC3 inhibitory toxin (APETx2) (see 2 Fig. S2B, Inset), which is known to inhibit ASIC3-type channels (32).

mRNA rela Human ASIC3a Channel Is a Sensor of Extracellular Alkalization. The 1 amplitude and direction of both the classic and the nonconventional sustained hASIC3a current, measured as the difference between the plateau current obtained at pH ranging 0 from 7.7 to 5.0 and the current level at the holding pH of 8.0 fi DRG Brain Spinal cord (Fig. 3A, Inset), were signi cantly different for pH values down to 6.6 (Fig. 3A). The nonconventional plateau currents were Fig. 1. Relative expression of ASIC mRNAs in human neuronal tissues. larger in amplitude and of opposite (i.e., positive) direction. This Quantitative RT-PCR experiments performed on total RNA from human DRG, suggested that the nonconventional hASIC3a current was con- brain, and spinal cord. (Inset) relative expression of ASIC3 in rat DRG and brain stitutively activated at pH 8.0; i.e., upon extracellular alkaliza- as a comparison. Expression is normalized to the expression of ASIC3 in DRG. tion. Indeed, the basal current level at pH 8.0 in cells presenting a nonconventional hASIC3a sustained current was significantly larger than in cells exhibiting the classic current (Fig. 3B). We found a high level of expression of hASIC3 mRNAs in spinal Moreover, APETx2 largely reduced the pH 8.0–induced resting cord and brain, comparable to the expression level in dorsal root current (Fig. 3B, Inset), strongly suggesting the participation ganglia (DRG), which is different from rodent where the ASIC3 of hASIC3a in this activity. We next measured the membrane mRNA is mainly restricted to DRG (Fig. 1, Inset). ASIC1 conductance (G) at different external pH values (Fig. 3C) mRNAs remain, however, the predominant ASIC transcripts in by applying 10-mV depolarizations to cells expressing the human brain (Fig. 1). nonconventional sustained current (Fig. 3C, Inset). Whole-cell membrane conductance significantly increased as the external Nonconventional Gating of hASIC3a. When heterologously expressed pH was shifted from 6.6 to 8.0, supporting the idea of an alkaline in F-11 cells, rat ASIC3 channel presented a typical biphasic in- pH-gated hASIC3a sustained current. Furthermore, the pH 8.0– fi ward current following moderate acidi cation (pH 8.0 to 7.0), with activated membrane conductance was fully abolished by APETx2 a transient phase followed by a noninactivating window current (Fig. 3C), demonstrating that hASIC3a carries an alkaline- (see Fig. S2A). In similar conditions, human ASIC3a showed two activated constitutive current in these cells. The hASIC3a alka- different types of currents (Fig. 2). We observed a “classic” ASIC3- line-induced sustained current is highly dependent on external type current in 46% of hASIC3-expressing cells (70 out of 153 Na+ ions (Fig. 3D). Indeed, the sustained current that developed cells, see Materials and Methods)(Fig.2,inset), which was related at pH 8.0 under normal sodium condition (145 Na) was almost to the typical current associated with ASIC3 (i.e., a transient phase suppressed when external sodium was replaced by N-Methyl + followed by a sustained plateau), and a “nonconventional” current, D-Glucamine (0 Na). This pH 8.0–induced Na -dependent cur- in 54% of cells (83 out of 153 cells) (Fig. 2), with an inward rent was inhibited by APETx2 (Fig. 3D). The I/V curve of the transient phase followed by an outward-going plateau phase more current sensitive to APETx2 (Fig. 3E,IAPETx2), obtained from positive than the current level at pH 8.0. Membrane capacitances a voltage ramp protocol on the different sustained current levels of the cells showing classic and nonconventional currents were recorded at pH 8.0 (Fig. 3D, Lower), showed that the pH 8.0– identical (30.2 ± 2.4 pF and 29.5 ± 1.6 pF, respectively; P = 0.81, induced hASIC3a current reversed at ∼33 mV (n = 10). A decrease in the external Na+ concentration from 145 to 45 mM led to a 30-mV shift (n = 4) of this reversal potential, which perfectly + hASIC3a corresponded to the shift of the Na ions reversal potential (theoretical ENa varying from ∼85 to ∼55 mV at 20 °C, according 8.0 7.0 8.0 to the Nernst equation). Although not as high as the classical acid-induced transient current (11), the human alkaline-sensitive 0 current thus exhibits a significant Na+ selectivity with a relative permeability of Na+ to K+ (PNa/PK) of 3.4. Thus, the human ASIC3a channel has the original property to 8.0 7.0 8.0 sense external pH in both directions (decrease and increase). It 600 pA can generate a transient activation upon acidification, and a sus- + 22s tained, mainly Na selective, current in response to alkalization. 600 pA non- Alkaline Sensitivity Is an Intrinsic Property of Human ASIC3. The al- 2s kaline-gated hASIC3a current was also observed in transfected convenonal classic COS-7 cells (Fig. 4A), injected Xenopus oocytes (Fig. 4B), as well as in transiently transfected mouse cortical neurons (Fig. 4C). Fig. 2. Macroscopic properties of the human ASIC3 currents. Whole-cell The characteristics of the alkaline-induced sustained current were recordings of hASIC3a currents performed at −80 mV from F-11 transfected cells. Currents were activated by rapid changes of the external pH from similar in the different expression systems (see SI Results). The pH 8.0 to 7.0, as indicated above each current trace. Dashed lines represent alkaline sensitivity of hASIC3a was steep, with maximal current at

the zero current level. The nonconventional current (main trace) and the pH 8.0, zero current level estimated around pH 6.5, and a Hill NEUROSCIENCE classic current (Inset) are represented. slope of 1.4 (Fig. 4D); i.e., smaller than that observed for the

Delaunay et al. PNAS | August 7, 2012 | vol. 109 | no. 32 | 13125 Downloaded by guest on September 30, 2021 when rat ASIC3 or human ASIC1a were expressed in F-11 cells A Isustained pH(test-8.0) * (Fig. 4E)orinXenopus oocytes (see Fig. S3F), strongly suggesting (pA) ** that this property was tightly associated with hASIC3. ASIC3 can * 81 100 ** classic form heteromeric channels with ASIC1a in rodents (32), and * 59 ** non- ** convenonal hASIC3 mRNAs are often coexpressed with hASIC1 transcripts 10 19 50 in human tissues (Fig. 1 and Fig. S1). We thus tested whether the 0 heteromeric channel also displayed the alkaline-sensitivity. All of 4 7 51 the oocytes expressing a heteromeric hASIC1a/hASIC3c channel 67 -50 8.0 7.0 8.0 (n = 7; see SI Materials and Methods) showed an alkaline-induced 0 sustained current in addition to the classical transient inward -100 Isst current (see Fig. S3 E and F). Both the sustained and the tran- -150 25 sient currents were blocked by APETx2 (500 nM) with a potency 29 (38%, P < 0.01 and 28%, P < 0.05, respectively) that is in good pHtest 7.7 7.4 7.0 6.6 5.0 agreement with the effects of the toxin already described on rat

B classic C 6.6 8.0 ASIC3-containing heteromeric channels (32). -70 mV non-convenonal -80 mV All together, these results show that the alkaline-sensitivity is 0 0pA an intrinsic and speciﬁc property of human ASIC3 channels. This 400 pA property is probably not associated with the C-terminal domain 5s

-200 APETx2 that differs between the three hASIC3 isoforms. 70 G (nS) *** 8 * 7 ** 5 **

-400

** 83 control * 6 AB * 5 hASIC3a, COS cells hASIC3a, Xenopus oocytes APETx2 +APETx2 5 -600 8.0 6.6 8.0 8.0 6.0 8.0 pH8.0 5 5 0 I basal 0 0 at pH8.0 100pA pH (pA) 2s 4 6.6 7.0 8.0 1nA 6.6 8.0 0 6.5 8.0 2s 50 nA 0 D +APETx2 E 1 - 212 (IAPETx2) Inorm 10 s pH 8.0 60 pA 1s 20 s 0Na 145Na 0,5 CDhASIC3a, mouse corcal neurons hASIC3a, F-11 cells 1 V (mV) -20 20 200 pA I/IpH8.0 -80 -60 -40 40 60 80 8.0 7.0 8.0 10 s ramp 2 1.0 protocol 0 80 -0,5 145 mM Na (mV) ext 0.8 45 mM Naext -50 -80 -1,0 0.6 8.5

6.8 0.4 pH6.6 Fig. 3. Properties of the nonconventional hASIC3a sustained current in F11 20 pA 0.2 cells. (A) Amplitudes of the hASIC3a sustained current component (Isst) for 2s 0 pH the classic (white bars) and nonconventional (black bars) currents recorded at 6.2 6.6 7.0 7.4 7.8 8.2 8.6 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, E 7.4 8.0 -30 -20 -10 0 10 20 30 Isst and 5.0) and the current level at the holding pH 8.0 (see Inset). The number of pH(8.0-7.4) experiment is indicated above each bar (**P < 0.01 and ***P < 0.001, Mann– hASIC3a (pA) 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 rASIC3 a Dunn’s post hoc test). As illustrated in the Inset, the APETx2 toxin at 1 μM 100 pA inhibited the basal current in cells expressing the nonconventional hASIC3a 2s current. (C) The macroscopic membrane conductances G, calculated from 10- hASIC1a 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 < Fig. 4. The alkaline-induced sustained current is independent of the ex- 0.05, **P < 0.01, and ***P < 0.001, repeated measures ANOVA test). (D) pression system. (A)pH6.6–evoked current recorded at −80 mV from COS Sensitivity of the pH 8.0–induced hASIC3a current to extracellular Na+ ions cells transfected with hASIC3a. The dotted line represents the zero current and APETx2 toxin (3 μM). The 0-Na condition was obtained by replacing ex- level, and the typical alkaline-induced sustained current is magniﬁed. + + ternal Na by N-Methyl D-Glucamine (NMDG ) ions. A voltage ramp protocol (B) Xenopus oocytes injected with hASIC3a also display a typical alkaline- was applied to the current levels obtained in the presence (1) and in the induced sustained current. The two current traces were recorded at −80 mV absence (2) of APETx2 (3 μM). E, I/V curve of the pH 8.0–induced APETx2- from the same oocyte. (C)pH7.0–evoked current recorded at −80 mV from sensitive hASIC3a current. 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 transient current activated by acidic pH (i.e., ∼3.0, Fig. S2B). from ﬁve different cells). Amplitudes of the sustained currents are normal- hASIC3b and hASIC3c, which generate classical acid-induced ized 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- currents with properties similar to hASIC3a when expressed in cell current recorded at −80 mV from F-11 cells expressing either hASIC3a, Xenopus oocytes, were also activated when the external pH is rASIC3, or hASIC1a channels. The alkaline-induced current was not observed shifted from 6.5 to 9 (see Fig. S3). The alkaline-induced current in rASIC3- or hASIC1a-expressing cells. rASIC3 showed a small widow current was blocked by amiloride (see Fig. S5A) and was not observed at pH 7.4 that was absent at pH 8.0.

13126 | www.pnas.org/cgi/doi/10.1073/pnas.1120350109 Delaunay et al. Downloaded by guest on September 30, 2021 Two Arginine Residues in the Extracellular Loop Are Involved in the similarly to what has been shown for two-pore domain alkali- Alkaline Sensitivity of Human ASIC3a Channel. Our data strongly activated K+ channel (33). Arginine 83 is located at the junction suggested that the sensitivity of hASIC3a to alkaline pH was di- between β1andβ2 linkers of the palm domain, a region that has rectly supported by the channel itself. Therefore, we looked at the recently been shown to control the sustained opening of ASIC1 structural elements involved in the alkaline sensitivity of the (34). Single (R68G or R83Q) or dual (RR68,83GQ) mutations of channel using a site-directed mutagenesis approach (Fig. 5). An those two arginine residues signiﬁcantly reduced the amplitude of ASIC3 chimeric channel containing rat ASIC3 transmembrane the alkaline-induced sustained current, compared with the wild- and intracellular domains and human ASIC3a extracellular loop type hASIC3a current (Fig. 5 C and D). The transient current showed alkaline sensitivity (Fig. 5A, Upper); whereas, a human amplitudes at pH5.0 were not different between the mutants and ASIC3a chimera containing the rat ASIC3 extracellular loop did the wild-type channels (Fig. 5E), showing a speciﬁc effect of the not (Fig. 5A, Lower). The human and rat ASIC3 extracellular two arginine residues on the alkaline-induced current. Introducing loops share a high percentage of homology (∼90%), and we fo- these two arginine residues within the extracellular loop of the rat cused our attention on two arginine residues (R68 and R83) that channel (GQ69,84RR mutant) induced a small alkaline sensitivity are only present in the extracellular loop of the human isoforms in 11 out of the 26 oocytes tested, although this mutant did not (Fig. 5B). The arginine 68 is located at the junction between the fully reconstitute the human properties (see Fig. S4A). These transmembrane domain 1 (TM1) and the extracellular loop, results demonstrate that the sustained activation of hASIC3a in response to extracellular alkalization is fully supported by the a critical hydrophobic location that suggested an unusual pK for a extracellular loop where the R68 and R83 residues are playing an this arginine, which could be involved in alkaline pH sensing, important role. Finally, it was possible to strongly reduce the acid sensitivity of the hASIC3 channel without affecting the alkaline-induced cur- A B rent (see Fig. S4B) by mutating two amino acids in the post-TM1 rASIC3-hLoop3 region that are crucial for H+ gating (35) (hASIC3-HH71,72NN). 6.6 8.0 Thus, our data suggest that it is possible to partially dissociate both functioning modes by mutating either arginines 68 and 83 or histidines 71 and 72. 50nA 10 s Alkaline Current of hASIC3a Is Modulated by Calcium, Lactic Acid, and hASIC3-rLoop3 Arachidonic Acid. The alkaline sensitivity of hASIC3a is only ob- 6.6 8.0 served in about half of the cells, suggesting the existence of regulatory mechanisms. We have evaluated the effect of several factors previously described that modulate rat ASIC3, including 50nA extracellular calcium, lactic acid, and arachidonic acid, on the 10 s alkaline sensitivity of hASIC3a. Extracellular calcium ions have

C hASIC3 hASIC3-R68G hASIC3-R83Q hASIC3-RR68,83GQ been involved in the gating mechanism of ASIC3 by protons (36). We thus tested whether the alkaline sensitivity of hASIC3a 6.6 8.0 6.6 8.0 6.6 8.0 6.6 8.0 was dependent on external calcium concentration. Changing the 0 extracellular calcium concentration, from normal (2 mM) to high (10 mM) or low (1 μM) levels, did not induce any alkaline sen- 100 nA 100 nA 100 nA 100 nA 10 s 10 s 10 s 100 s sitivity in cells only displaying the conventional hASIC3a current; i.e., not sensitive to external alkaline pH (n = 5). However, the basal hASIC3a current recorded at pH 8.0 from cells displaying the alkaline sensitivity was modulated by changes of external D E calcium concentration (Fig. 6A). It was increased in low calcium Sustained current Transient current conditions whereas it was reduced at high calcium concen- pH(6.6-8.0), nA pH(5.0-8.0), nA trations, similarly to what was previously described for acid-in- 80 53 -800 46 36 duced rat ASIC3 current (36). Lactic acid, a potentiator of the * 53 17 60 ** -600 rat ASIC3 current, which acts by lowering the concentration of 46 2+ 2+ * Ca and Mg ions (17), also increased the alkaline-induced 40 ** * -400 36 ** 17 hASIC3a current (Fig. 6B). 20 -200 Arachidonic acid (AA) is a well-known mediator of inﬂammation, which potentiates rat ASIC3 current (16, 19, 36). Over all of the 0 0 hASIC3a-expressing F-11 cells tested (n = 37), 14 initially displayed an alkaline-induced sustained current when external pH was switched from 7.0 to 8.0 (see Materials and Methods). This alkaline- Fig. 5. Mapping of the structural elements involved in the alkaline sensi- sensitive current was always potentiated by AA, as indicated by the tivity of hASIC3. (A) Effect of external alkalization (from pH 6.6 to 8.0) on effect on the basal current recorded at pH 8.0 (Fig. 6C, Left). The oocytes expressing the rat ASIC3 chimera containing the extracellular loop of effect of AA was also observed in Xenopus oocytes injected with hASIC3a; (Upper) rASIC3-hLoop3 or the human ASIC3a chimera containing hASIC3a (Fig. S5B), but was absent in F-11 cells transfected with rat the extracellular loop of rASIC3; (Lower) hASIC3-rLoop3. (B) Schematic rep- ASIC3 (Fig. 6C, Right). It is interesting to note that AA alone was resentation of two ASIC subunits in a functional channel. The two arginines sufﬁcient to trigger a current at physiological pH 7.4 from cells dis- only present in human ASIC3, and not found in rat, are indicated. Adapted playing the alkaline sensitivity (Fig. 6D), and this AA-induced by permission from ref. 4 (Copyright 2007, Macmillan Publishers Ltd). (C) hASIC3a current was inhibited by APETx2 (Fig.6D, Inset). More- 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, over, AA was also able to induce, or unmask, the alkaline sensitivity one-way ANOVA followed by Tukey’s post hoc test). (E) Bar graph of the in 10 of the 23 remaining cells (43%) that initially did not display

amplitudes of the pH 5.0–induced transient currents measured from oocytes an alkaline-induced current in response to a pH switch from 7.0 to NEUROSCIENCE expressing the wild-type or the arginine mutants described in C and D. 8.0 (Fig. 6E). Arachidonic acid, calcium, and lactic acid are thus

Delaunay et al. PNAS | August 7, 2012 | vol. 109 | no. 32 | 13127 Downloaded by guest on September 30, 2021 A B ASIC1a-type currents (37). This does not preclude an expression 2mM 10mM 1μM 2+ lactate of ASIC3 in other brain regions or neuronal subpopulations. 0 [Ca ]ext 7.0 8.0 We demonstrate that all of the hASIC3 variants (i.e., hASIC3a, 7 hASIC3b, and hASIC3c) have an original biophysical property -200 that makes them capable to sense extracellular pH variations in 7 both directions; i.e., acidiﬁcation and alkalization. This original -400 property never described for ASICs is supported by two distinct functioning modes of hASIC3, a classical transient mode in re- -600 sponse to pH decrease, combined with a sustained mode in 7 Ibasal ** 50 pA response to pH increase (see Fig. S6). The alkaline sensitivity * at pH8.0 (pA) 2s appears to be a speciﬁc and intrinsic property of hASIC3 chan- + C D nels, and seems to be different, for instance, from the NH4 - hASIC3a rASIC3

7.4 7.4 +AA 0 0 induced ASIC currents described by Pidoplichko and colleagues +AA (38). This property can be potentiated and /or induced by one or 200 “ ” 13 APETx2 more molecular switches, such as arachidonic acid, which is 13 -200 5 able to induce an hASIC3 current at physiological resting pH, and 400 17 9 it may be associated with particular physiological or pathological 600 control conditions in the peripheral and central nervous system. + AA -400 Sensitivity to alkalization is shared by other excitatory ion Ibasal 17 50 pA 9 at pH8.0(pA) *** 40 s (pA) ** channels expressed in the sensory pathway, such as transient receptor potential vanilloid 1 (TRPV1) and transient receptor po- E +AA tential ankyrin 1 (TRPA1) (39, 40). TRPV1, like hASIC3, is activated by acidic pH in addition to alkaline pH (39). However, 7.0 8.0 7.0 8.0 7.0 8.0 7.0 8.0 7.0 8.0 7.0 8.0 7.0 8.0 contrary to hASIC3, neither TRPV1 nor TRPA1 are directly activated by extracellular alkalization. They are activated by the secondary intracellular alkalization that follows extracellular application of high-pH solution. Accordingly, human ASIC3 directly senses extracellular alkalization via two arginine residues in its 100 pA extracellular domain; R68, which is only present in human and 4 s primates, and R83, also found in a few other species, while TRPV1 and TRPA1 need residues in the N-terminal cytoplasmic Fig. 6. Modulation of the hASIC3a alkaline-sensitive current. Whole-cell patch-clamp experiments performed on transfected F-11 cells clamped at domain (a histidine and two cysteines, respectively). This makes −80 mV. (A) Effect of different external calcium concentrations on the basal hASIC3 very sensitive to extracellular alkalization, with a maxi- sustained current recorded at pH8.0 in cells transfected with hASIC3a. The mal sustained depolarizing current reached around pH 8.0 and concentration of 1 μM free calcium was obtained by combining 5 mM EGTA a Hill slope of 1.4. This channel appears therefore as a unique and 4.97 mM CaCl2 at pH 8.0, as calculated with the maxchelator software and very efficient sensor of extracellular acidification or alkalin- (*P < 0.05 and **P < 0.01, Friedman test followed by a Dunn’s post hoc test). ization near the resting physiological pH. (B) Effect of lactate (20 mM) on the pH 8.0–induced hASIC3a sustained Although it remains to be established whether acidic- and alka- – μ current. (C) Bar graph showing the potentiating effect of AA (10 20 M) on line-pH activations of hASIC3 produce action potentials in human the basal hASIC3a current recorded at pH 8.0 in cells displaying the alkaline neurons, it is however possible to speculate, based on the experi- 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 ments done in rodent neurons, that the fast and large depolarization also represented (Right). (D) External application of AA (20 μM) was suffi- generated by a rapid drop in extracellular pH will most probably cient to trigger a hASIC3a current at physiological pH 7.4 in cells displaying lead to the generation of action potentials. On the other hand, the the alkaline sensitivity. This AA-induced current was inhibited by APETx2 alkaline-activated current, which has slower kinetics and smaller (5 μM, Inset). (E) Representative current trace showing the effect of arach- amplitudes, will generate a long-lasting depolarization that would idonic acid (10 μM) on a cell that initially displayed no pH 8.0–induced sus- significantly modulate neuron excitability in response to other tained current. stimuli (sensitization). How neurons expressing hASIC3 can dis- tinguish and integrate the different signals associated with external acidification or alkalization? This clearly depends on the presence modulators of the hASIC3a alkaline-sensitive current, with a par- or absence of the alkaline sensitivity, and on the amplitude, di- ticularly potent effect of AA. rection, and kinetics of the pH change within the tissue (see Discussion SI Discussion). It is also important to consider that the hASIC3 channel could fulfill different roles in different neuron populations In humans, ASIC3 has three splice variants and little is known and/or in different physiological conditions. This is further sup- about the molecular and biophysical properties of these channels. ported by the fact that the alkaline sensitivity is not always expressed We found here that the ASIC3a variant represents the main but can be induced by factors such as AA and probably others that ASIC3 mRNA in the human nervous system. The ASIC3c tran- remain to be identified. script is also present in nervous tissues but at a much lower level, In sensory neurons, the human ASIC3 channels could therefore and ASIC3b is barely detected. Our data show the presence of contribute to pain perception associated with both tissue acidosis, ASIC3 transcripts in human brain and spinal cord, which rein- as in rodents, and tissue alkalization. Tissue alkalization, although forces the idea that, conversely to the rat, the expression of hu- less frequent than acidification, occurs in several physiological or man ASIC3 is not restricted to peripheral sensory neurons (10). pathophysiological situations including for instance hyperventila- These results suggest that hASIC3 may play a role in central tion, which produces peripheral nerves hyperexcitability as pC02 neurophysiological functions although ASIC1 transcripts remain decline, and leads to paraesthesia (41), or the effusion of alkaline largely predominant in human brain. It is difficult to know to what pancreatic fluids in patients having a pancreatic-pleural fistula that extent hASIC channels are functionally expressed in central often causes chest pain (42). neurons. The only study of ASIC currents performed in cultured Human ASIC3 can thus constitute a particularly efficient mo- human cortical neurons exclusively reported the presence of lecular sensor for alkalization and/or recovery from acidification in

13128 | www.pnas.org/cgi/doi/10.1073/pnas.1120350109 Delaunay et al. Downloaded by guest on September 30, 2021 neurons and other cell types where it may be expressed. Unveiling were made at room temperature using an axopatch 200B ampliﬁer (Axon the biological conditions that control the alkaline sensitivity of this Instruments) with a 3-kHz low-pass ﬁlter (Krohn-Hite). Data were sampled at channel will certainly help to better understand the physiological 10 kHz, digitized by a Digidata 1440 A-D/D-A converter (Axon Instruments) and recorded on a hard disc using pClamp software (version 10; Axon consequences of this remarkable property. Instruments). Patch pipettes (1–4MΩ) contained (in millimolars): 135 KCl, 2.5 Na -ATP, 2 MgCl , 2.1 CaCl , 5 EGTA, 10 Hepes (pH 7.25 with KOH). The Materials and Methods 2 2 2 control bath solution contained (in millimolars): 145 NaCl, 5 KCl, 2 MgCl2, F-11 and COS Cells Culture and Transfection. The F-11 cell line was grown as 2 CaCl2, 10 Hepes, 10 glucose (pH 7.4 with NaOH). Mes was used instead of described previously (16). One day after plating, cells were transfected with Hepes to buffer the solution pH, which ranged from 6 to 5 and ASIC currents either pIRES2-hASIC3a-HcRed, pIRES2-hASIC3a-EGFP, pIRES2-hASIC3b-EGFP, were induced by shifting one out of eight outlets of the microperfusion pIRES2-hASIC3c-EGFP, pIRES2-hASIC1-EGFP, or pIRES2-rASIC3-EGFP vectors using system from a holding control solution (i.e., pH 7.4 or 8.0) to an acidic (pH < the JetPEI reagent according to the supplier’s protocol (Polyplus Transfection 7.4) or an alkaline (pH > 7.4) test solution. Glucose (10 mM) was added to the SA). Fluorescent cells were used for patch-clamp recordings 2–4 d after trans- control bath solution. fection, and cells were considered as “hASIC3-positive” only when the transient

current amplitude was at least 400 pA at pH 6.6 and/or 1 nA at pH 5.0 (i.e., IpH5.0 ACKNOWLEDGMENTS. We thank M. Lazdunski for fruitful discussion and

> 400 pA and/or IpH5.0 > 1 nA). Among these cells, we considered that an alkaline comments on the manuscript; A. Baron, S. Diochot, P. Inquimbert, and sensitivity was present only when the amplitude of the sustained current in- M. Christin for helpful discussion; and C. Chevance for secretarial assistance. We thank the Fondation pour la Recherche Medicale (FRM); the Association duced upon alkalization (pH 8.0) was of at least 10 pA (i.e., I > 10 pA). pH8.0 Française contre les Myopathies (AFM); the Agence Nationale de la Recherche (ANR); the Fédération pour la recherche sur le cerveau (FRC); and FIS PI08/ Patch-Clamp Experiments. We used the whole-cell conﬁguration of the patch- 0014, FIS PI11/01601, RD07/0062/0006 (Inst. Salud Carlos III, Spain), and clamp technique to measure membrane currents (voltage clamp). Recordings 2009SGR869 (Gen. Catalunya) for ﬁnancial support.

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