A glial DEG/ENaC channel functions with neuronal channel DEG-1 to mediate specific sensory functions in C. elegans

Abstract

Mammalian neuronal DEG/ENaC channels known as ASICs (acid-sensing ion channels) mediate sensory perception and memory formation. ASICS are closed at rest and are gated by protons. Members of the DEG/ENaC family expressed in epithelial tissues are called ENaCs and mediate Na(+) transport across epithelia. ENaCs exhibit constitutive activity and strict Na(+) selectivity. We report here the analysis of the first DEG/ENaC in Caenorhabditis elegans with functional features of ENaCs that is involved in sensory perception. ACD-1 (acid-sensitive channel, degenerin-like) is constitutively open and impermeable to Ca(2+), yet it is required with neuronal DEG/ENaC channel DEG-1 for acid avoidance and chemotaxis to the amino acid lysine. Surprisingly, we document that ACD-1 is required in glia rather than neurons to orchestrate sensory perception. We also report that ACD-1 is inhibited by extracellular and intracellular acidification and, based on the analysis of an acid-hypersensitive ACD-1 mutant, we propose a mechanism of action of ACD-1 in sensory responses based on its sensitivity to protons. Our findings suggest that channels with ACD-1 features may be expressed in mammalian glia and have important functions in controlling neuronal function.

Figure 1

The C. elegans DEG/ENaC channel ACD-1 (acid-sensitive channel degenerin-like) shares similarity with mammalian BLINaC and hINaC. (A) We designated C24G7.2 as ACD-1 because of its sensitivity to acidic solutions. C24G7.2 protein sequence and alignments with hINaC (29% identity and 45% similarity) and mouse and rat BLINaC (28% identity and 44% similarity). The transmembrane domains TM1 and TM2 and the region of the protein deleted in acd-1(bz90) knockout are indicated by the grey boxes and black line, respectively. The transmembrane domains were identified by threading ACD-1 sequence onto the sequence of chicken ASIC1a (Jasti et al, 2007), using PyMOL (www.PyMOL.org). The box and the skull designate position S513 in ACD-1 corresponding to the amino acid that when mutated to Val or Thr in MEC-4 induces hyperactivation of the channel (A713V or T, the (d) mutation). The dotted boxes indicate potential glycosylation sites. Alignment by ClustalW (available at bioweb.pasteur.fr). (B) A schematic diagram representing ACD-1 predicted topology. The potential glycosylation sites are indicated by the branches. The circles depict cysteine residues; a region just preceding TM2 is rich in cysteines. This feature is conserved in DEG/ENaC subunits across species (Benos and Stanton, 1999). (C) Dendrogram of C. elegans (bold), Drosophila (RPK and PPK, underlined)), Helix apersa (FaNaC, underlined) and mammalian (italic) DEG/ENaC channel subunits. ACD-1 subunit has a grey background.

Figure 2

Properties of wild-type and mutated ACD-1 channel. (A) Typical Na⁺ currents in an oocyte injected with acd-1 cRNA, perfused with a physiological NaCl solution. Voltage steps were from −160 to +100 mV from a holding potential of −30 mV. (B) Current–voltage relationships for oocytes injected with acd-1 cRNA when the oocytes are perfused with a physiological NaCl solution (open circles, n=15) and with a physiological saline plus 500 μM of DEG/ENaC channel blocker amiloride (filled circles, n=14). (C) Selectivity of ACD-1 channels to monovalent cations, established by determining the ratio of the inward current at −160 mV when perfusing the oocytes with NaCl, LiCl and KCl (n=8). (D) Typical Na⁺ currents in an oocyte injected with acd-1(S513V) cDNA, perfused with a physiological NaCl solution. Voltage steps were from −160 to +100 mV from a holding potential of −30 mV. The (S513V) mutation in ACD-1 is analogous to A713V in MEC-4(d) (Driscoll and Chalfie, 1991). (E) We perfused the same oocyte shown in (D) with a solution in which we substituted NaCl with CaCl₂. Note that perfusing the oocytes with the CaCl₂ solution does not activate the oocyte endogenous Ca²⁺-activated Cl⁻ current that we have used in the past as a measure of MEC-4(d) permeability to Ca²⁺ (Bianchi et al, 2004). Similar results were obtained for wild-type ACD-1 channels. (F) S513V mutation does not hyperactivate ACD-1. Average current amplitude at −160 mV from oocytes injected with wild-type and S513V mutant ACD-1, n=5. The averages are not significantly different by t-test (P=0.12). (G) Amiloride dose–response curves for ACD-1 and ACD-1(S513V) (K_i=99 and 17 μM, respectively, n=5 for both). Data are expressed as mean±s.e. (H) Lack of voltage dependence of amiloride blockade in ACD-1 channels (n=5). (I) The (S513V) mutation in ACD-1 introduces voltage dependence of amiloride blockade in ACD-1 channels, suggesting that amino acid 513 is located within the membrane electric field (n=5). Data points were fitted with a Woodhull model (=0.14). Data are expressed as mean±s.e.

Figure 3

ACD-1∷GFP is expressed in the glial amphid sheath cells where it is needed for acid avoidance behaviour. (A, B) DIC and fluorescent (GFP channel) micrographs of a transgenic animal expressing Ex[ACD-1∷GFP;VAP-1∷RFP] showing the expression of ACD-1∷GFP in two large cells in the head of the worm (the other cell is on another focal plane) whose cell bodies are on either side of the pharyngeal bulb. vap-1 encodes for a venom-allergen-like-protein expressed in amphid sheath cells (Perens and Shaham, 2005). (C) The same animal was photographed using the rhodamine filter. This revealed overlapping of green (ACD-1∷GFP) and red (VAP-1∷RFP) signals (see (D), merged), establishing that ACD-1∷GFP is expressed in amphid sheath cells. (E–H) DIC and fluorescent micrographs taken with GFP and rhodamine filters of the tip of the nose of the same animal shown in (A–D), showing the expression of ACD-1∷GFP in amphid sheath cell processes (only one is shown here, the other one is on another focal plane). Front is left. (I, J) C. elegans exhibit an amiloride-sensitive aversive behaviour towards acidic solutions. (I) Average avoidance index to solutions in which pH was adjusted to 3.5 using acetic acid (CH₃COOH) or HCl, measured using our drop test assay (see Materials and methods). For experiments in which we added amiloride, we preincubated animals in 1 mM amiloride for 30 min and then assayed adding amiloride to the pH 3.5 solution. Number of experiments performed was 8, 5 and 3, respectively. In each experiment, at least 10 animals were tested. (J) Quantification of ratio of animals responding (grey circles) at different pHs for wild-type C. elegans, deg-1;acd-1 mutants expressing wild-type ACD-1, and deg-1;acd-1 mutants expressing ACD-1(D419N). ACD-1 expression was driven in the glial amphid sheath cells by the vap-1 promoter (Perens and Shaham, 2005). We fitted the wild-type C. elegans pH/avoidance curve by sigmoidal equation, which gave pH_0.5 values of 3.8. Data are expressed as mean±s.e. n of trials was 6–9 at pH 3.5 and 4.5 and 4–11 at the other pHs; at least 10 animals in each trial were tested. At least two transgenic lines were generated per construct and no statistical difference was found between data obtained from these lines. (K) ACD-1 functions with DEG-1 to mediate acid sensation. Ratio of animals responding to M13 solution buffered to pH 3.5 with acetic acid for wild-type, deg-1, acd-1, deg-1;acd-1 double mutants and deg-1;acd-1 double mutants expressing exogenous ACD-1 cDNA under the control, of acd-1 promoter, sra-6 promoter (Troemel et al, 1999) and glial-specific vap-1 and T02B11.3 promoters (Perens and Shaham, 2005) and (Supplementary Figure 2). ACD-1 expression in sheath cells but not ASH neurons rescues the acid insensitivity of deg-1;acd-1 mutants. ACD-1 construct used for the determining ACD-1 expression pattern, which includes acd-1 promoter and genomic sequence (B, F), also rescued acid avoidance defect of deg-1;acd-1 mutants but did not enhance acid avoidance when expressed in wild-type C. elegans. The figure also shows that a construct containing the deg-1 promoter that we have used for our expression pattern studies (Figure 4A) and deg-1 cDNA sequence (Pdeg-1∷DEG-1) rescues the acid avoidance deficit of deg-1;acd-1 mutant. Number of experiments was from 4 to 8 with at least 10 animals tested each. Data are expressed as mean±s.e. ^**, ^*P<0.01 and 0.05 by comparison with wild-type (WT) animals by t-test, respectively. NS stands for nonsignificant difference between the averages indicated by the red bracket by t-test.

Figure 4

DEG/ENaC channels ACD-1 and DEG-1 are also needed for lysine chemotaxis. (A) DEG-1 is expressed in ASK and ASG chemosensory neurons. Merged photographs of a transgenic C. elegans expressing DEG-1∷GFP stained with lipophylic dye DiO. Overlapping of green and red fluorescence identifies ASK sensory neurons as one of the neurons that expresses DEG-1∷GFP. Another nearby neuron that expresses DEG-1∷GFP was tentatively identified as ASG. DiO staining (red) is also evident in ADF and ASI sensory neurons. Dorsal view, front to the left. (B) Chemotaxis index towards lysine acetate was determined for wild-type, deg-1, acd-1 mutants and deg-1;acd-1 double mutant, deg-1;acd-1 double mutants expressing exogenous ACD-1 in amphid glia and deg-1;acd-1 double mutants expressing DEG-1 in sensory neurons. ACD-1 expression was under the control of glial-specific vap-1 promoter (Perens and Shaham, 2005), whereas DEG-1 expression was under its native promoter (see Materials and methods). The compromised chemotaxis of deg-1;acd-1 to lysine is rescued by the exogenous expression of ACD-1 in the glial amphid sheath cells and DEG-1 in sensory neurons. Number of trials was 5–14, with at least 30 animals assayed in each trial. (C) The chemotaxis index of wild-type (dark grey) and deg-1;acd-1 double mutant animals (light grey) was determined at different concentrations of lysine acetate (0.25, 0.5 and 1 M). n=4–11. (D) We determined the chemotaxis index to Na acetate for wild-type (dark grey) and deg-1;acd-1 double mutant animals (light grey) using different concentrations of Na acetate (0.2, 0.4 and 0.8 M). n=4–11. Data are expressed as mean±s.e. ^**, ^*P<0.01 and 0.05 by comparison with wild-type (WT) animals by t-test, respectively.

Figure 5

DEDG/ENaC channel ACD-1 exhibits a strong sensitivity to acidic solutions. (A) Example of currents elicited by voltage steps from −160 to +100 mV from a holding potential of −30 mV in a Xenopus oocyte injected with acd-1 cRNA and exposed to a physiological NaCl saline solution at pH 7.2. (B) The same oocyte was exposed to a solution at pH 5.5, which resulted in the inhibition of ACD-1 currents. (C) When we returned to physiological pH 7.2, ACD-1 currents returned to control levels. (D) ACD-1 currents are equally suppressed by extracellular solutions buffered with acetic acid and HCl and are reduced by incubation with 20 mM extracellular Na acetate and lysine acetate, suggesting that both extracellular and intracellular acidification cause channel inhibition (Thomas, 1984; Zampighi et al, 1988). For incubation with 20 mM Na acetate and 20 mM lysine acetate, we incubated oocytes in the NaCl solution in which we added Na acetate or lysine acetate for 1 h at room temperature. Control oocytes were incubated under the same conditions in NaCl physiological saline. Oocytes were then transferred to control NaCl solution for 15 min prior to recordings. We could not recover currents to control levels after 3 h of wash in Na acetate- and lysine acetate-treated oocytes. This is not unusual and it has been observed by others using this technique for intracellular acidification (Thomas, 1984; Zampighi et al, 1988). n was 8 for each bar. (E) Sensitivity of wild-type ACD-1 and ACD-1(D419N) channels to extracellular pH. Currents were recorded at -160 mV, normalized to maximal currents and best fitted with the Hill's equation (K_i=pH 6.4, n=0.9 for wild-type ACD-1; K_i for ACD-1(D419N) was 7.3). n is 4–27. Data are expressed as mean±s.e. ^**Indicates P<0.01 by comparison with untreated oocytes by t-test.