FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1904902384> ?p ?o ?g. }

• W1904902384 endingPage "1863" @default.
• W1904902384 startingPage "1852" @default.
• W1904902384 abstract "Article22 March 2011free access Two types of chloride transporters are required for GABAA receptor-mediated inhibition in C. elegans Andrew Bellemer Andrew Bellemer Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USAPresent address: Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA Search for more papers by this author Taku Hirata Taku Hirata Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Michael F Romero Michael F Romero Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Michael R Koelle Corresponding Author Michael R Koelle Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Andrew Bellemer Andrew Bellemer Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USAPresent address: Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA Search for more papers by this author Taku Hirata Taku Hirata Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Michael F Romero Michael F Romero Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN, USA Search for more papers by this author Michael R Koelle Corresponding Author Michael R Koelle Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Author Information Andrew Bellemer1, Taku Hirata2, Michael F Romero2 and Michael R Koelle 1 1Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT, USA 2Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN, USA *Corresponding author. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, SHM CE30, New Haven, CT 06520-8024, USA. Tel.: +1 203 737 2271; Fax: +1 203 785 6404; E-mail: [email protected] The EMBO Journal (2011)30:1852-1863https://doi.org/10.1038/emboj.2011.83 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Chloride influx through GABA-gated Cl− channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl− gradient established in part by K+–Cl− cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na+-driven Cl−–HCO3− exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K+–Cl− cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl− extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl− gradient such that Cl− flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl− gradient. Our results show that Na+-driven Cl−–HCO3− exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain. Introduction GABA is the primary inhibitory neurotransmitter in the mammalian brain (Mody et al, 1994). Cl− influx through GABAA receptors results in neuronal hyperpolarization and inhibition. This response depends on maintenance of a relatively low intracellular Cl− concentration, resulting in a Cl− reversal potential more negative than the cell's resting membrane potential (Farrant and Kaila, 2007). Many immature neurons maintain high intracellular Cl− levels, resulting in a Cl− reversal potential more positive than the cell's resting potential, so that GABA can elicit Cl− efflux and an excitatory response (Ben-Ari, 2002; Blaesse et al, 2009). Thus, neurons regulate intracellular Cl− levels to control the magnitude and polarity of the effects of GABA, and this in turn has been shown to powerfully affect neural development and activity (Fiumelli et al, 2005; Akerman and Cline, 2006). Understanding this type of ‘ionic plasticity’ (Rivera et al, 2005) requires understanding which transporters control intracellular Cl− levels. Multiple transporters move Cl− across the plasma membrane. The most extensively studied are the Cl−-extruding K+–Cl− cotransporter KCC2 and the Cl−-accumulating Na+–K+–2Cl− cotransporter NKCC1 (Blaesse et al, 2009). The excitatory effect of GABA on immature hippocampal and cortical neurons is due in part to their low KCC2 expression and high NKCC1 expression. GABA becomes inhibitory as neurons upregulate expression of KCC2 and downregulate expression of NKCC1 (Rivera et al, 1999; Yamada et al, 2004; Akerman and Cline, 2006). However, KCC2 is apparently not the sole Cl− extruder (Rivera et al, 1999; Gulacsi et al, 2003). Na+-driven Cl−–HCO3− exchangers (also known as Na+-driven anion exchangers, or NDAEs) can extrude Cl− and could contribute to the cellular Cl− gradient and hyperpolarizing GABA action (Gulacsi et al, 2003; Kim and Trussell, 2009). However, to date no specific transporter of this type has been shown to have such a role. The SLC4 family of ion transporters includes multiple Cl−–HCO3− exchangers (Romero et al, 2004). Among these, human and squid NDCBE and Drosophila NDAE1 have been shown to function as NDAEs that extrude Cl− (Romero et al, 2000; Grichtchenko et al, 2001; Virkki et al, 2003). Despite expression of NDCBE in mammalian neurons and its characterized role in regulating neuronal intracellular pH (Schwiening and Boron, 1994; Grichtchenko et al, 2001; Chen et al, 2008a, 2008b), its role in regulating neuronal intracellular Cl− homeostasis has not been fully investigated. The nematode Caenorhabditis elegans has been developed as a model organism for genetic analysis of GABAergic neurotransmission (Jorgensen, 2005). C. elegans possesses 19 GABAergic motor neurons that synapse on the body wall muscles (BWMs) to promote hyperpolarization and lengthening of muscles contralateral to contracting muscles, producing sinusoidal body bends and coordinated locomotion (McIntire et al, 1993b). UNC-49 is the C. elegans GABAA receptor homologue (Bamber et al, 1999; Richmond and Jorgensen, 1999). UNC-49 function is required in the BWMs to generate coordinated body bends (McIntire et al, 1993a, 1993b; Bamber et al, 1999). More recently, C. elegans has been established as a model system in which to identify Cl− transporters that control GABA signalling. C. elegans egg-laying behaviour can be blocked by inactivating the hermaphrodite-specific neurons (HSNs) with a mutation in the putative transmembrane receptor EGL-47 (Moresco and Koelle, 2004). Suppressor mutations that reactivate the HSNs to allow egg laying to occur identify genes required for inhibitory signalling by GABA, including the gene encoding the K+–Cl− cotransporter KCC-2 (Tanis et al, 2009). In this work, we have used this screening strategy to identify the Na+-driven Cl−–HCO3− exchanger ABTS-1 and show that it functions partially redundantly with KCC-2 to control GABAergic signalling in C. elegans neurons and muscles. Results Mutations in abts-1 suppress egl-47(dm) to reactivate egg laying Wild-type animals retain 13.1±0.9 eggs in utero (Figure 1A), while egl-47(dm) mutants fail to lay eggs and thus retain a far larger number, 57.5±2.7 (Figure 1B). More strikingly, wild-type young adults lay many eggs through the first ∼12 h of adulthood (70.5±4.4 eggs per pool of five animals assayed), while we have never observed a young adult egl-47(dm) animal lay an egg (Figure 1D). Thus, egl-47(dm) animals provided an exquisitely clean background in which we were able to identify suppressor mutations that restore HSN activity to reactivate egg laying. Figure 1.Mutations in abts-1 suppress the egl-47(dm) egg-laying defect. (A–C) Representative images of wild-type, egl-47(dm), and abts-1(ok1566); egl-47(dm) worms. The average number of unlaid eggs is shown (±95% confidence interval). n⩾30 animals for each genotype. Eggs with eight or fewer cells (arrowheads) and eggs with more than eight cells (arrows). (D) Eggs laid by pools of five young adult worms, by 18 h after the late-L4 stage. Wild-type young adults lay many eggs, while egl-47(dm) young adults never lay any eggs. Mutations in abts-1 significantly suppress the egl-47(dm) egg-laying defect (P<0.005; comparing bars with white asterisks to the black asterisked control with zero eggs). For each genotype n=6 pools of five worms each. Error bars in this and subsequent figures represent standard errors except when otherwise noted. (E) Proportion of eggs laid at an early developmental stage. Wild-type (bar 1), egl-47(dm) (bar 2), abts-1(vs145) (bar 3), abts-1(ok1566) (bar 5), and kcc-2(vs132) (bar 7) animals all lay a low proportion of early-stage eggs that have not yet reached the eight-cell stage. abts-1(vs145); egl-47(dm) (bar 4), abts-1(ok1566); egl-47(dm) (bar 6), and kcc-2(vs132); egl-47(dm) (bar 8) animals all lay a high proportion of such early-stage eggs (asterisks indicate P<0.05 compared with the bar 2 control). n⩾100 eggs for each genotype, error bars show the 95% confidence interval. These results show that egl-47(dm) significantly stimulates egg laying in the absence of ABTS-1 or KCC-2. Download figure Download PowerPoint We used the egl-47(dm) suppressor screen to identify the vs145 suppressor mutation, which strongly reactivated egg laying in an egl-47(dm) background (Figure 1C and D). We mapped and cloned vs145 and discovered a mutation in the anion bicarbonate transporter gene, abts-1, which encodes a protein similar to mammalian HCO3− transporters of the SLC4 family (Sherman et al, 2005). The vs145 mutation results in the substitution of an arginine for a glycine residue within a predicted transmembrane helix (Figure 3A). We obtained a deletion mutation ok1566 that is a putative null since it removes sequences encoding all of one and parts of two other predicted transmembrane helices. Because vs145 and ok1566 are phenotypically similar (Figure 1D and E; data not shown), vs145 appears to be a strong loss-of-function mutation. Both the vs145 and ok1566 alleles restored egg laying in egl-47(dm) young adults. The double mutants abts-1(vs145); egl-47(dm) and abts-1(ok1566); egl-47(dm) (Figure 1C) retained only 7.3±1.2 and 7.3±0.8 eggs in utero, respectively, compared with the 57.5±2.7 eggs retained by egl-47(dm) single mutants (Figure 1B). As young adults, the double mutants abts-1(vs145); egl-47(dm) and abts-1(ok1566); egl-47(dm) laid 20.3±4.0 and 30.2±4.5, respectively, compared with the 0.0±0.0 eggs laid by egl-47(dm) single mutants (Figure 1D). Thus, mutations in abts-1 suppress egl-47(dm) by reactivating egg laying in young adults. Loss of abts-1 causes the egl-47(dm) mutation to stimulate rather than inhibit egg laying abts-1 mutations are similar to kcc-2 mutations in that both show the same remarkable interaction with egl-47(dm). While egl-47(dm) strongly inhibits egg laying in a wild-type genetic background, it instead stimulates egg laying in a kcc-2 mutant background such that kcc-2; egl-47(dm) mutants lay eggs at a much higher rate than do wild-type animals (Tanis et al, 2009). The stimulatory effect of the egl-47(dm) mutation on function of HSNs lacking KCC-2 may be analogous to the excitatory action of GABA on immature mammalian neurons that do not express KCC2 (Rivera et al, 1999). To assay for an increased rate of egg laying, we measured the developmental stage of freshly laid eggs. C. elegans eggs are fertilized internally and undergo cell divisions in utero prior to being laid. In wild-type animals only ∼5% of freshly laid eggs are at an early developmental stage (defined as eight cells or fewer). Mutants that lay eggs at a higher rate than do wild-type animals retain eggs in utero for a shorter period of time and thus lay a higher percentage of their eggs at an early developmental stage (Chase and Koelle, 2004). Using this assay, we found that while wild-type animals, egl-47(dm) mutants, and abts-1 single mutants lay a low percentage of early-stage eggs, abts-1; egl-47(dm) double mutants lay over 60% of their eggs at an early developmental stage (Figure 1E). Thus, abts-1 mutations, like kcc-2 mutations, reverse the effect of egl-47(dm) on egg laying, but do not change egg-laying behaviour in the absence of egl-47(dm) or other challenges to the egg-laying system. abts-1 mutants lay fewer cumulative eggs than do wild-type animals (Figures 1C and 4), but this observation can be attributed to decreased egg production in abts-1 mutants (Supplementary Figure S1), an effect that is also observed in kcc-2 mutants (Tanis et al, 2009). ABTS-1 is a Na+-driven chloride–bicarbonate exchanger To analyse ABTS-1 function, we used Xenopus oocytes to express the ABTS-1 protein and monitored membrane ion transport using intracellular ion-selective microelectrodes (Figure 2). Previous experiments with the ABTS-1 ‘B’ isoform (isoforms are shown in Figure 3A) revealed that it functioned as an electroneutral Na+/HCO3− cotransporter (Romero and Boron, 1998). However, this early study did not address the potential role of Cl−, as the transport activity was quite low. Studies of the ABTS-1 ‘A’ isoform indicated that it functions as a Cl−–HCO3− exchanger, but did not address any potential role of Na+ (Sherman et al, 2005). In the present study, we sought to more fully characterize the transport properties of ABTS-1, specifically to determine whether it is an NDAE that acts to extrude Cl−. Figure 2.ABTS-1 is a sodium-driven chloride–bicarbonate exchanger. (A–C) Electrophysiology data from pH microelectrodes in Xenopus oocytes. In the presence of 1.5% CO2/10 mM HCO3− (pH 7.5), both ABTS-1 (A) and Drosophila NDAE1 (B) responded to Cl− replacement (0Cl−) by increasing pHi (HCO3− influx to oocyte), while water-injected oocytes (C) showed no response. By contrast, Na+ removal (0 Na+) resulted in dramatic pHi decreases (HCO3− efflux from oocyte) in oocytes expressing ABTS-1 (A) and NDAE1 (B), while water-injected oocytes (C) showed no response. Rates of alkalinization during Cl− and Na+ removal (# × 10−5 pH units/s) are indicated above the respective segments of the pHi traces. (D) Electrophysiology data from halide microelectrodes in Xenopus oocytes. In oocytes expressing ABTS-1 and water-injected controls, replacement of extracellular Cl− with gluconate produced no change in [Cl−]. When extracellular Cl− was replaced by I−, the electrode sensed I− influx in oocytes expressing ABTS-1, but not in water-injected controls. (E) Models illustrating the suggested transport activity of ABTS-1 and NDAE1 in response to removal of extracellular Cl− (a) and in response to removal of extracellular Na+ or addition of extracellular I− (b). The transport activity illustrated in panel (Ea) is also the predicted activity of ABTS-1 under normal conditions. The stoichiometry shown, with two HCO3− ions transported, is consistent with the observed electroneutrality of the transport. Download figure Download PowerPoint Figure 3.ABTS-1 is expressed predominantly in neurons and muscles. (A) The gene structure of abts-1. Two abts-1 transcripts, abts-1a and abts-1b, have different start sites that splice onto common exons. Black bars represent exons and connecting lines indicate introns. Indicated are the polyadenylation site (AAA) and the SL1 trans splice leader (SL1). Also displayed are the transmembrane domain coding region (grey bar) and the locations of the vs145 point mutation and the ok1566 deletion mutation. (B–D) GFP fluorescence in animals carrying an abts-1a promoter::gfp::abts-1 3′ UTR reporter transgene. This reporter transgene was expressed in many cells including the BWMs, pharyngeal muscles, and head and tail neurons. (E–G) GFP fluorescence in animals carrying an abts-1b promoter::gfp::abts-1 3′ UTR reporter transgene. This reporter transgene was expressed primarily in neurons, including the HSN, and in the vulval muscles. Scale bars in all images are 50 μm. Download figure Download PowerPoint We found that the transport activity of ABTS-1 is consistent with Na+-driven Cl−–HCO3− exchange. The ion transport activity by ABTS-1 is somewhat lower than that of many other HCO3− transporters (note differences in the magnitude of pH change in Figure 2Ab and Bb), complicating some measures of its transport activity. We therefore tested the hypothesis that ABTS-1 is an NDAE by directly comparing ABTS-1 activity (Figure 2A) to that of a known Na+-driven Cl−–HCO3− exchanger, Drosophila NDAE1 (Figure 2B). Water-injected control oocytes had minimal background transport activity (Figure 2C). ABTS-1 (Figure 2A) and NDAE1 (Figure 2B) expression resulted in HCO3− transport (measured with intracellular pH microelectrodes) in response to removal of Cl− or Na+. With Cl− removal (0 Cl−) both ABTS-1 and NDAE1 produced an increase of intracellular pH (pHi), indicating HCO3− movement into the cell (model in Figure 2Ea). This response is characteristic of Cl−–HCO3− exchange (Romero et al, 2000; Chang et al, 2009). Next, we removed Na+ (0 Na+). Both ABTS-1 and NDAE1 responded by decreasing pHi, indicating HCO3− movement out of the cell (model in Figure 2Eb). This effect is consistent with Na+-driven Cl−–HCO3− exchange (Romero et al, 2000; Grichtchenko et al, 2001). Monitoring of Vm indicated that this transport activity was electroneutral (Supplementary Figures S2 and S3). Many Cl− transport systems will also transport other halides (e.g. I− and Br−). The order of preference of halides (the Hoffmeister series) may be used to distinguish anion transport systems (Wright and Diamond, 1977). Figure 2D shows that ABTS-1 expression provided Xenopus oocytes with I− transport activity. By calibrating our anion-sensitive microelectrodes (see Materials and methods), we determined that for ABTS-1-expressing oocytes incubated in high extracellular [I−], intracellular [I−] reached ∼250 μM within 5 minutes. In contrast, intracellular [I−] did not increase in control oocytes injected with water rather than ABTS-1 copy RNA (cRNA) (Figure 2D). The model in Figure 2Eb represents the proposed transport mechanism that occurs during Na+ removal (Figure 2A) and during I− addition (Figure 2D): ABTS-1 transports Na+ and HCO3− out of the cell while transporting halides into the cell. ABTS-1 is broadly expressed in C. elegans neurons and muscles C. elegans KCC-2 is expressed in excitable cells such as neurons and muscles where it presumably extrudes Cl− to support inhibitory neurotransmission by ligand-gated Cl− channels (Tanis et al, 2009). We observed a similar pattern of ABTS-1 expression. To examine the expression pattern of ABTS-1, we constructed abts-1 promoter::gfp::abts-1 3′ UTR reporter transgenes in which GFP expression was driven by 5′ promoter and 3′ untranslated region sequences. Because the available ABTS-1 cDNAs showed the presence of two transcripts with different 5′ exons (Figure 3A), we generated two reporter constructs using the promoter regions for each of the two isoforms (Supplementary Figure S4). We found that the abts-1a promoter drove expression of GFP in head, tail, and lateral neurons as well as in BWMs, pharyngeal muscles, and neural support cells (Figure 3B–D). The abts-1b promoter drove expression of GFP in head neurons, tail neurons, and the HSNs, as well as weak expression in the vulval muscles (Figure 3E–G). ABTS-1 is required for UNC-49-mediated inhibition of egg laying Given the apparently similar mutant phenotypes, Cl− extrusion activities, and expression patterns of ABTS-1 and KCC-2, we hypothesized that ABTS-1 may have a similar role to KCC-2 in establishing the cellular Cl− gradient that supports the inhibitory action of GABA-gated Cl− channels. UNC-49 is the C. elegans GABAA receptor homologue, and its function is dependent on KCC-2 (Bamber et al, 1999; Tanis et al, 2009). The GABAA receptor agonist muscimol is a potent inhibitor of egg laying in wild-type animals, but not in animals that have compromised UNC-49 function. Thus, muscimol does not inhibit egg laying in animals lacking UNC-49 or in animals lacking KCC-2 (Tanis et al, 2009). We found that muscimol also fails to inhibit egg laying in abts-1 mutants. In control wild-type animals, muscimol inhibited egg laying by 97%: over 2 h, pools of five animals laid 83.5±5.6 eggs in the absence but only 2.7±0.9 eggs in the presence of muscimol (Figure 4A). This effect requires UNC-49 and KCC-2, since the corresponding mutants showed only 11 and 12% inhibition of egg laying by muscimol, respectively (Figure 4A). Similarly, we found that the abts-1 mutant showed only 12% inhibition of egg laying by muscimol (Figure 4A). Thus, ABTS-1 may function similarly to KCC-2 in generating the Cl− gradient required for muscimol to inhibit egg laying via UNC-49. Figure 4.ABTS-1 is required for the inhibition of egg-laying behaviour by muscimol. The average number of eggs laid by pools of five animals over 2 h in the presence or absence of 0.5 mM muscimol. unc-49, kcc-2, and abts-1 mutants were all resistant to the inhibitory effects of muscimol on egg-laying behaviour. Brackets indicate the percent inhibition by muscimol. For each genotype n=6 pools of five worms each. Download figure Download PowerPoint Inhibition of egg laying by muscimol requires KCC-2 function in the HSNs (Tanis et al, 2009). We found that ABTS-1 function is also required in the HSNs for normal sensitivity to muscimol. The inhibitory effect of muscimol on egg laying was partially restored by re-expression of ABTS-1 in the HSNs of abts-1 mutants as compared with abts-1 mutants expressing a GFP control protein (Supplementary Figure S5). The fact that re-expression of ABTS-1 only partially rescued muscimol sensitivity may indicate that ABTS-1 function is required in an additional cell type or that our transgene drives expression of ABTS-1 in the HSNs at a sub-wild-type level. We also observed that re-expression of ABTS-1 in the HSNs of the abts-1; egl-47(dm) mutant rescued the egl-47(dm) egg-laying defect (Supplementary Figure S6), providing additional evidence for ABTS-1 function in the HSNs. UNC-49 activation excites rather than inhibits BWMs in the absence of ABTS-1 While GABA is normally an inhibitory neurotransmitter, neurons that do not have a mechanism to extrude Cl− may have relatively high intracellular Cl− levels that lead to an excitatory effect of GABAA receptor activation (Kaneko et al, 2004). Such an effect is observed in C. elegans BWMs, which receive inputs from GABAergic motor neurons that act to inhibit and lengthen the BWMs during the animal's locomotion (McIntire et al, 1993b). While wild-type animals exposed to muscimol undergo simultaneous relaxation of all BWMs and thus an increase in total body length, kcc-2 mutants experience muscle contraction and shortening of total body length (Tanis et al, 2009). The standard electrophysiological method for assaying changes in Cl− reversal potential (Blaesse et al, 2009) appears not to work on C. elegans muscle cells due to the failure of the ionophore gramicidin to perforate the membranes of these cells (data not shown). Nevertheless, the behavioural response to muscimol strongly suggest that there is a depolarizing shift in the Cl− reversal potential in the muscles of kcc-2 mutants, resulting in an excitatory effect of UNC-49 activation. We found that abts-1 mutants exposed to muscimol undergo a decrease in body length similar to that observed in kcc-2 mutants. Wild-type animals exposed to muscimol experienced a significant increase in body length from 1207±6 μm before exposure to 1277±9 μm after exposure (Figure 5A, B, and E). In contrast, the abts-1 mutant underwent a significant decrease in body length when exposed to muscimol from 1021±7 μm before exposure to 984±8 μm following exposure (Figure 5C–E). This shortening effect was comparable to that observed in kcc-2 mutants (Figure 5E). Both the lengthening observed in wild-type animals and the shortening observed in abts-1 and kcc-2 mutants were dependent on the UNC-49, as loss of UNC-49 function in any genetic background resulted in no change in body length following muscimol exposure (Figure 5E). Figure 5.ABTS-1 is required for the inhibition of muscle contraction by muscimol. (A, B) Images of representative wild-type animals in the absence (A) or presence (B) of 1 mM muscimol. A line was drawn down the centre of each worm image as indicated to measure body length. Average lengths±standard errors are shown for each condition. n⩾30 animals for each genotype. Scale bars=100 μm. (C, D) Images of representative abts-1 animals in the absence (C) or presence (D) of 1 mM muscimol. (E) Average body length before and after 2 h of exposure to 1 mM muscimol. Wild-type animals displayed significant increases in body length following muscimol exposure, while abts-1 and kcc-2 mutants displayed significant decreases. Strains with unc-49 mutations showed no changes in body length. For each genotype, body-length measurements were normalized to body length prior to muscimol exposure. n⩾30 animals for each genotype. Asterisks indicate significant changes with P<0.005. (F) Average body length of transgenic animals before and after 2 h of exposure to 1 mM muscimol. A muscle-specific promoter was used to transgenically express cDNAs encoding the control protein GFP or ABTS-1. Re-expression of ABTS-1 in the BWMs of abts-1 mutants caused a significant increase in body length following muscimol exposure, in contrast to the muscimol-induced shortening observed in abts-1 mutants expressing GFP in the BWMs. n⩾50 animals for each genotype. Asterisks are as in panel (E). (G) Model depicting that Cl−-extruding transporters establish a gradient of Cl− ions across the membrane such that the Cl− reversal potential is more negative than the membrane resting potential. Thus, UNC-49 activation leads to Cl− influx and hyperpolarization. (H) Model depicting that cells not expressing Cl−-extruding transporters maintain relatively high intracellular Cl− levels such that the Cl− reversal potential is more positive than the resting potential. Thus, UNC-49 activation leads to Cl− efflux and depolarization. Download figure Download PowerPoint KCC-2 and UNC-49 both function in the BWMs to mediate the effect of muscimol on body length (Tanis et al, 2009), and we found that ABTS-1 also functions in the muscles to control this effect of muscimol. Wild-type and abts-1 mutant animals expressing the control protein GFP in their BWMs showed lengthening and shortening in response to muscimol, respectively, comparable to that observed in non-transgenic wild-type and abts-1 mutant animals (Figure 5F). Re-expression of ABTS-1 in the muscles of abts-1 mutants converted their muscimol response from shortening back to a lengthening similar to that seen in the wild-type controls (Figure 5F). These results are consistent with a model in which ABTS-1 acts as a Cl− extruder in the BWMs, along with KCC-2, to help set the cellular Cl− gradient required for UNC-49-mediated inhibition (Figure 5G) and muscle lengthening. In this model, loss of ABTS-1 or KCC-2 function results in accumulation of intracellular Cl− and thus a depolarizing shift in the Cl− reversal potential to produce UNC-49-mediated excitation and muscle contraction (Figure 5H). ABTS-1 and KCC-2 function redundantly in the BWMs to mediate endogenous inhibitory GABA signalling Mutants that lack UNC-49 display sluggish and uncoordinated locomotion (McIntire et al, 1993a; Bamber et al, 1999). Despite the fact that abts-1 and kcc-2 mutants show excitatory rather than inhibitory responses to the GABAA agonist muscimol, these mutants still show well-coordinated locomotion (Supplementary Videos S1, S2, S3, and S4). One model suggested by these observations is that modest changes in the cellular Cl− gradient produced by loss of one transporter do reverse the flow of Cl− through the UNC-49 Cl− channel, but the low levels of UNC-49 activation caused by endogenous, synaptically released GABA still result in ‘shunting’ inhibition of the muscles to allow coordinated movement (see the Discussion for further elaboration of this model). The model that ABTS-1 and KCC-2 act in parallel predicts that a mutant lacking both ABTS-1 and KCC-2 would show phenotypic defects more severe than the relatively mild defects observed in abts-1 or kcc-2 single mutants. We found that the abts-1; kcc-2 double mutant indeed has severe defects in muscle contractility and locomotion not observed in either single mutant. We measured the body length of animals not exposed to any drug and found that wild-type body length was 1212±6 μm, and the body lengths of abts-1 and kcc-2 mutants were modestly shorter at 1064±8 μm and 1145±6 μm respectively (Figure 6A, B, C, and E). The unc-49 mutant was also modestly shorter than" @default.
• W1904902384 created "2016-06-24" @default.
• W1904902384 creator A5010116834 @default.
• W1904902384 creator A5017249033 @default.
• W1904902384 creator A5034690690 @default.
• W1904902384 creator A5041914018 @default.
• W1904902384 date "2011-03-22" @default.
• W1904902384 modified "2023-09-27" @default.
• W1904902384 title "Two types of chloride transporters are required for GABA_Areceptor-mediated inhibition in<i>C. elegans</i>" @default.
• W1904902384 cites W1539985358 @default.
• W1904902384 cites W1540119902 @default.
• W1904902384 cites W1550426196 @default.
• W1904902384 cites W1554045794 @default.
• W1904902384 cites W1586193028 @default.
• W1904902384 cites W1588629441 @default.
• W1904902384 cites W1605667399 @default.
• W1904902384 cites W1647075334 @default.
• W1904902384 cites W1915376905 @default.
• W1904902384 cites W1965190304 @default.
• W1904902384 cites W1971126847 @default.
• W1904902384 cites W1972816943 @default.
• W1904902384 cites W1974231491 @default.
• W1904902384 cites W1981839201 @default.
• W1904902384 cites W1986830025 @default.
• W1904902384 cites W1991917470 @default.
• W1904902384 cites W1992321658 @default.
• W1904902384 cites W1992962615 @default.
• W1904902384 cites W1996064510 @default.
• W1904902384 cites W2008785965 @default.
• W1904902384 cites W2009214699 @default.
• W1904902384 cites W2023373036 @default.
• W1904902384 cites W2024328209 @default.
• W1904902384 cites W2025946976 @default.
• W1904902384 cites W2032291923 @default.
• W1904902384 cites W2039469985 @default.
• W1904902384 cites W2039637448 @default.
• W1904902384 cites W2041357157 @default.
• W1904902384 cites W2047293798 @default.
• W1904902384 cites W2058202542 @default.
• W1904902384 cites W2059067548 @default.
• W1904902384 cites W2064363302 @default.
• W1904902384 cites W2074821443 @default.
• W1904902384 cites W2077793836 @default.
• W1904902384 cites W2078613709 @default.
• W1904902384 cites W2084712021 @default.
• W1904902384 cites W2091745271 @default.
• W1904902384 cites W2095758052 @default.
• W1904902384 cites W2097961247 @default.
• W1904902384 cites W2110475552 @default.
• W1904902384 cites W2113188978 @default.
• W1904902384 cites W2120336502 @default.
• W1904902384 cites W2122163057 @default.
• W1904902384 cites W2124697178 @default.
• W1904902384 cites W2129296423 @default.
• W1904902384 cites W2130630814 @default.
• W1904902384 cites W2132818618 @default.
• W1904902384 cites W2134820311 @default.
• W1904902384 cites W2136779789 @default.
• W1904902384 cites W2155011915 @default.
• W1904902384 cites W2161121393 @default.
• W1904902384 cites W2165654778 @default.
• W1904902384 cites W4246813496 @default.
• W1904902384 cites W4296759707 @default.
• W1904902384 doi "https://doi.org/10.1038/emboj.2011.83" @default.
• W1904902384 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3101993" @default.
• W1904902384 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21427702" @default.
• W1904902384 hasPublicationYear "2011" @default.
• W1904902384 type Work @default.
• W1904902384 sameAs 1904902384 @default.
• W1904902384 citedByCount "32" @default.
• W1904902384 countsByYear W19049023842012 @default.
• W1904902384 countsByYear W19049023842013 @default.
• W1904902384 countsByYear W19049023842014 @default.
• W1904902384 countsByYear W19049023842015 @default.
• W1904902384 countsByYear W19049023842016 @default.
• W1904902384 countsByYear W19049023842017 @default.
• W1904902384 countsByYear W19049023842018 @default.
• W1904902384 countsByYear W19049023842019 @default.
• W1904902384 countsByYear W19049023842021 @default.
• W1904902384 countsByYear W19049023842023 @default.
• W1904902384 crossrefType "journal-article" @default.
• W1904902384 hasAuthorship W1904902384A5010116834 @default.
• W1904902384 hasAuthorship W1904902384A5017249033 @default.
• W1904902384 hasAuthorship W1904902384A5034690690 @default.
• W1904902384 hasAuthorship W1904902384A5041914018 @default.
• W1904902384 hasBestOaLocation W19049023842 @default.
• W1904902384 hasConcept C104317684 @default.
• W1904902384 hasConcept C149011108 @default.
• W1904902384 hasConcept C168258287 @default.
• W1904902384 hasConcept C170493617 @default.
• W1904902384 hasConcept C191897082 @default.
• W1904902384 hasConcept C192562407 @default.
• W1904902384 hasConcept C2776674594 @default.
• W1904902384 hasConcept C2777771985 @default.
• W1904902384 hasConcept C2778695967 @default.
• W1904902384 hasConcept C2778944004 @default.
• W1904902384 hasConcept C55493867 @default.
• W1904902384 hasConcept C86803240 @default.

• next