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Muscle contraction is normally mediated by the release of neurotransmitters from motor neurons. Here we demonstrate that protons can act as a direct transmitter from intestinal cells to stimulate muscle contraction. During the C. elegans defecation motor program the posterior body muscles contract even in the absence of neuronal inputs or vesicular neurotransmission. In this study, we demonstrate that the space between the intestine and the muscle is acidified just prior to muscle contraction and that the release of caged protons is sufficient to induce muscle contraction. PBO-4 is a putative Na+/H+ ion exchanger expressed on the basolateral membrane of the intestine, juxtaposed to the posterior body muscles. In pbo-4 mutants the extracellular space is not acidified and the muscles fail to contract. The pbo-5 and pbo-6 genes encode subunits of a ‘cys-loop’ proton-gated cation channel required for muscles to respond to acidification. In heterologous expression assays the PBO receptor is half-maximally activated at a pH of 6.8. The identification of the mechanisms for release and reception of proton signals establishes a highly unusual mechanism for intercellular communication.
The release of small molecule transmitters is the major mechanism of fast information exchange in the central nervous system. Usually, classical neurotransmitters are stored in synaptic vesicles. Calcium stimulates the fusion of the synaptic vesicles with the plasma membrane and releases the neurotransmitter into the synaptic cleft. The transmitter then binds to ligand-gated ion channels that either excite or inhibit the target cell. However, other neurotransmitters are released via non-canonical mechanisms. For example, gaseous neurotransmitters are not released via synaptic vesicles, but pass directly through membranes (Baranano et al., 2001). These unusual properties concealed the functions of gaseous neurotransmitters for quite some time. There are other candidate molecules that may function as bona fide neurotransmitters, but have been set aside due to our incomplete understanding of their biological functions. The proton in particular has properties that make it well suited to play a role in neurotransmission.
Although sparse, hydrogen ions (H+) are of enormous biological significance (Kaila and Ransom, 1998). For example, free H+ ions can ionize the side groups of proteins and thereby affect the structure and function of proteins. Recently it has been demonstrated that molecules involved in proton signaling are vital for appropriate central nervous system function. Specifically, mouse knockouts have demonstrated that proteins involved in both proton secretion and sensing are required for broad physiological functions that range from pH homeostasis to learning and memory (Bell et al., 1999; Denker et al., 2000; Wemmie et al., 2002; Zha et al., 2006).
To function as an intercellular signal, H+ ions must be released from a cell in a regulated way. Na+/H+ exchangers (NHEs) are ubiquitously expressed proteins capable of regulated release of protons. NHEs catalyze the electroneutral exchange of Na+ and H+ ions. In general, eukaryotic NHEs transport one Na+ ion in and one H+ ion out of the cell, thereby alkalinizing the cytoplasm and acidifying the extracellular environment. NHEs are regulated by many distinct signaling molecules including calmodulin (CaM), phosphatidylinositol 4,5-bisphosphate (PIP2) and calcineurin homologous protein (CHP) (Aharonovitz et al., 2000; Lin and Barber, 1996; Pang et al., 2001; Wakabayashi et al., 1994; Wakabayashi et al., 1997). NHEs have been implicated in numerous physiological processes such as acidification of the intestinal lumen, intracellular pH homeostasis, cell volume regulation, and reabsorption of NaCl across epithelial cells (Counillon and Pouyssegur, 2000; Orlowski, 1993). In addition, some phenotypes suggest that proton secretion may play broader roles in cellular function. For example, NHE1 mutant mice exhibit growth retardation, ataxia, seizures and defects in cell morphology and adhesion (Bell et al., 1999; Denker et al., 2000). However, it is unclear whether these phenotypes are due to a loss of proton-mediated intercellular signaling or to a side effect of pH misregulation.
If H+ ion secretion mediates signals between cells, there must be proton receptors. It is known that protons can modulate neurotransmission. Practically all ligand-gated ion channels are affected by extracellular pH shifts. Ionotropic acetylcholine and NMDA receptors are reversibly inhibited by acidic pH and potentiated by alkaline pH (Del Castillo et al., 1962; Giffard et al., 1990; Palma et al., 1991; Traynelis and Cull-Candy, 1990). Conversely, ionotropic GABA receptors are potentiated by acidic pH and inhibited by alkaline pH (Kaila, 1994; Smart and Constanti, 1982; Takeuchi and Takeuchi, 1967). In addition to playing a modulatory role in neurotransmission, protons have been demonstrated to be the major ligand for particular classes of ligand-gated ion channels. Acid-sensing ion channels (ASICs) are members of the ENaC/Degenerin family of ion channels and are gated by protons (Kaila and Ransom, 1998; Waldmann and Lazdunski, 1998). Some of these channels are involved in monitoring pH in external environments and may act in nociceptive sensory neurons (Krishtal, 2003). Recently, a ‘cys-loop’ family proton-gated ion channel was identified in cyanobacteria that could be involved in adaptation to environmental pH (Bocquet et al., 2007). It is possible that some pH receptors sense internally generated pH changes, since a subset of ASIC receptors are expressed in the central nervous system and mouse mutants lacking ASIC1 exhibit locomotory and dendritic spine defects (Wemmie et al., 2002; Zha et al., 2006). However, it is unclear if protons are released in a physiologically relevant manner to mediate intercellular communication.
Here we demonstrate that H+ ions act as a transmitter for muscle contraction during the defecation cycle in the nematode Caenorhabditis elegans. The defecation cycle is a stereotyped behavior that occurs every 50 seconds for the life of the animal and is characterized by the coordinated activation of three independent muscle contractions (Croll, 1975; Thomas, 1990). The defecation motor program begins by a sustained contraction of the posterior body muscles, these muscles relax, three seconds later the anterior body muscles contract and a moment later the enteric muscles contract, expelling the contents of the intestine (Figure 1A). The intestine itself is the timekeeper for the defecation cycle (Dal Santo et al., 1999). The cycle period is set by the activity of the inositol trisphosphate (IP3) receptor which regulates release of calcium from storage organelles in the intestine. An anteriorly-directed calcium wave directly precedes the motor program and controls the dynamics of the posterior body muscle contraction (Espelt et al., 2005; Norman et al., 2005; Peters et al., 2007).
How might the activity in the posterior intestine direct the contraction of the posterior body muscles? Neither extensive laser ablations of neurons nor mutants in synaptic transmission disrupted the posterior body contraction, suggesting that signaling for this muscle contraction appears to be controlled via a non-neuronal mechanism. To identify the genes required for signaling posterior body contractions, we screened for mutants defective for this motor step (Pbo mutants). In this study, we describe the pbo-4, pbo-5, and pbo-6 genes. PBO-4 is most closely related to the mammalian Na+/H+ exchanger NHE1 and is localized to the posterior intestine. PBO-5 and PBO-6 encode subunits of a proton-gated ‘cys-loop’ cation channel. These studies suggest that H+ ions act as a primary transmitter that activates the posterior body contraction during the defecation cycle in C. elegans.
Calcium is released from intracellular stores in the intestinal cells immediately before the posterior body contraction of the defection cycle, suggesting that this calcium spike might initiate the behavioral program (Dal Santo et al., 1999). During locomotion, all body muscles are stimulated by acetylcholine motor neurons. As expected, mutants lacking acetylcholine synthesis (cha-1 choline acetyltransferase) are paralyzed, however these animals exhibit perfectly normal posterior body contractions (posterior body contractions/cycle: ChAT cha-1(b401ts) 42/43). Furthermore, mutations that disrupt synaptic vesicle fusion (unc-13) or dense core vesicle fusion (unc-31/CAPS) result in animals that are also paralyzed but still have normal posterior body contractions (unc-13(s69) 46/49; CAPS unc-31(e928) 87/87). Finally extensive laser ablations failed to identify any neuron that contributed to the initiation of posterior body contraction, suggesting that this behavior is mediated via a non-neuronal mechanism (E.M. Jorgensen, H.R. Horvitz, unpublished).
To identify components of the signaling pathway required for posterior body contraction, we screened for mutants defective in this motor step. Recessive mutations in two genes, pbo-4 and pbo-5, specifically eliminate this step in the motor program (Figure 1B and C). Importantly, locomotion is normal in both pbo-4 and pbo-5 mutants. Because locomotion and posterior body contraction rely on the same muscles, the lack of muscle contraction during the defecation cycle in pbo-4 and pbo-5 mutants is not a result of degeneration or a developmental defect in the posterior body muscles.
The pbo-4(n2658) allele was mapped by standard two and three factor mapping and the pbo-4 gene identified by microinjection rescue. The cosmid K09C8 rescued the phenotype of n2658 and ok583 mutants, and an 8.5 kb subclone fragment containing only the K09C8.1 open reading frame was sufficient to rescue the posterior body contraction defect in ok583 mutants (Figure S1A). To confirm the identity of pbo-4, we sequenced genomic DNA from mutants and found mutations within the K09C8.1 open reading frame in each pbo-4 allele (Figure S1A and Table S1). The K09C8.1 gene is also known as nhx-7 (Nehrke and Melvin, 2002), but for clarity we refer to it as pbo-4.
The cDNA of pbo-4 encodes a predicted 783 amino acid protein (Figure S2) that is homologous to Na+/H+ exchangers. Plasma membrane Na+/H+ ion exchangers mediate the electroneutral exchange of one Na+ ion into the cell for one H+ ion out of the cell, and thus increase intracellular pH while acidifying the extracellular space. Na+/H+ ion exchangers are multidomain proteins containing an intracellular N-terminal domain, 12 transmembrane domains, and an intracellular carboxy terminal tail (Figure S1B and S2) (Wakabayashi et al., 2000). NHEs can be subdivided into separate families and are distinguished by their subcellular localization and regulatory mechanisms. PBO-4 shares the highest sequence identity with NHE1 (26% identity), and conversely, the closest homolog to NHE1 in the C. elegans genome is PBO-4. PBO-4 also shares relatively high sequence identity with NHE2 and NHE3 (22% and 21% respectively). The carboxy terminal domain is not well conserved among the diverse members of the Na+/H+ ion exchanger family, and each member contains distinct modulatory sites that greatly affect transport activity. For example, the carboxy terminus of NHE1 contains PIP2 binding domains, a calmodulin (CaM) binding domain and numerous phosphorylation sites; whereas, NHE3 lacks the PIP2 and calmodulin binding domains (Orlowski and Grinstein, 2004). Similar to NHE1, the carboxy tail of PBO-4 contains a predicted PIP2 binding domain, a calmodulin (CaM)-binding domain, and CamKII and PKC phosphorylation sites (Figure S2).
The defect in the posterior body contractions in mutants suggested that PBO-4 would be expressed in either the posterior body muscles, where the defect occurs, or in the intestine, where the defecation clock resides (Dal Santo et al., 1999). We generated a transgene with superecliptic pHluorin GFP (pHluorin) inserted into the N-terminal domain of PBO-4 and expressed it in pbo-4(ok583) mutants. pHluorin is a pH-sensitive form of GFP whose fluorescence is quenched by acidic media (Miesenbock et al., 1998) and the superecliptic form is a version of pHluorin with enhanced pH sensitivity (Sankaranarayanan and Ryan, 2000). The pHluorin::PBO-4 fusion protein rescued the posterior body contraction defect of the mutants, demonstrating that the protein was functional and properly localized (Figure 1B). The fluorescently-tagged protein was expressed on the basolateral surface of the intestine, exclusively in the most posterior intestinal cells (Figure 2A), consistent with previous observations (Nehrke and Melvin, 2002). To confirm basolateral expression, we exposed the posterior intestine in situ -- without exposing the apical, luminal surface -- to acidic media which quenched the majority of the pHluorin::PBO-4 fluorescence (Figure S3). The expression pattern of PBO-4 demonstrates that the protein resides on the basolateral surface of the intestine, appropriately localized to release a signal from the intestine to the posterior body wall muscles. Electron micrographs of the posterior intestine-muscle interface demonstrate that muscle arms extend from the posterior muscles and form intestino-muscular junctions (Figure 2B).
To further demonstrate that PBO-4 functions in the intestine, we rescued pbo-4(n2658) with the pHluorin::PBO-4 fusion driven by the promoter from the vitellogenin gene vit-2, which is expressed in all intestinal cells. Vitellogenin is synthesized in the intestine and transported to oocytes, where it serves to provide essential nutrients to the developing embryo. Because vitellogenins are only required in mature oocytes, the vit-2 promoter is expressed only in adult stages. We found that L4 animals were not rescued by this construct, while one-day-old adults were almost fully rescued (Figure 3). This result demonstrates that PBO-4 is required solely in intestinal cells, and that the pbo-4 defect is due to an acute loss of signaling function, not due to a developmental defect in the intestine.
Since the posterior body contraction is not mediated by classical neurotransmitters or peptides it is possible that acidification of the coelomic space by PBO-4 may stimulate contraction of the posterior body muscles. To visualize such a signal, we used superecliptic pHluorin, a pH-sensitive GFP whose fluorescence exhibits a pKa of pH 7.1 and is quenched at low pH (Miesenbock et al., 1998; Sankaranarayanan and Ryan, 2000). The superecliptic pHluorin was inserted into the first extracellular loop of PBO-4 (pHluorin::PBO-4) to monitor extracellular pH (Figure S1B and S2). If dissected intestines were exposed to acidic media, fluorescence was rapidly quenched demonstrating that the pH sensor was on the surface (Figure S3). To determine whether the posterior coelomic space was acidified in vivo prior to a posterior body contraction, we glued worms to agar but left the tip of the tail free to observe muscle contractions. Rapid fluorescence quenching always preceded the posterior body contraction (Figure 4A,D and Movie S1). Fluorescence spikes were characterized by a steep rise to peak (τ=1.9 ± 0.09 s; n=18 recordings from separate animals; 10−90% of peak), and a slower exponential decay to baseline fluorescence (τ=2.9 ± 0.3 s n=18) and preceded the muscle contraction with a latency of approximately 2 seconds (Figure 4E-H).
To test whether PBO-4 is fully required for the acidification, we deleted the C-terminal tail of the pHluorin::PBO-4 transgene to generate a non-rescuing fusion (pHluorin::PBO-4(ΔC)). This truncated construct expressed in the posterior intestinal cells, but there were very few, if any, fluorescence transients. In five of fourteen animals with the truncated construct no transients were detected. The remaining nine animals possessed significantly reduced transients [ΔF/F=5.0 ± 0.7%, n=18 for pHluorin::PBO-4 vs. ΔF/F =1.0 ± 0.2%, n=14 for truncated pHluorin::PBO-4(ΔC), P<0.0001] that were quantitatively and qualitatively distinct from the rescuing pHluorin reporter (Figure 4B, E-H and Movie S2). The small changes in pH remaining in this strain could represent residual activity of the truncated PBO-4 protein or activity from another Na+/H+ ion exchanger such as NHX-6 which is also expressed in the intestine (Nehrke and Melvin, 2002). Thus, PBO-4 is required for the transient acidification of the extracellular space that precedes posterior body contractions.
To test whether the fluorescent events were merely artifacts produced by the posterior body contractions, the reporter construct was introduced into the pbo-5(ox4) mutant that fails to execute posterior body contractions. In pbo-5 mutants, fluorescence transients were detected with similar frequency, amplitude, and kinetics as in the wild-type background, indicating that fluorescence transients cannot be attributed to posterior body contraction motion (Figure 4C, E-H).
Because pbo-5 mutants retain the pH transient but lack a posterior body contraction, it is likely that the pbo-5 gene product acts downstream of PBO-4, possibly as part of the pH sensing pathway in the muscle. The pbo-5 gene was mapped to a small region on the right arm of chromosome V by standard two and three factor mapping (Figure S4A). The posterior body contraction defect observed in pbo-5(n2303) mutants was rescued by injection of a yeast artificial chromosome (YAC) Y44A6 containing C. elegans genomic DNA from this region. To further localize the rescuing activity, a series of deletions were made from the Y44A6 YAC and injected into pbo-5(n2303) animals (Figure S4A). In addition, we analyzed the breakpoints of several chromosome V terminal deletions. From these experiments rescuing activity was localized to a single predicted open reading frame, Y44A6E.1 (Figure S4B). To demonstrate pbo-5 was encoded by Y44A6E.1, we constructed a mini-gene composed of genomic DNA containing 4.2 kb of promoter sequences and exons 1 and 2; exon 2 was fused to the rest of the open reading frame using sequences from a cDNA. Animals carrying this plasmid were fully rescued for posterior body contraction (Figure 1C, ox4; oxEx[PBO-5]). Additionally, we sequenced twenty pbo-5 alleles and found mutations within the Y44A6E.1 open reading frame in all of them (Figure S4C, Table S2). The pbo-5 mutations fall into four categories: (1) nine terminal deletions, (2) three nonsense mutations, (3) seven missense mutations, and (4) one splice junction mutation (Table S2). The predicted pbo-5 cDNA encodes a 504 amino acid protein.
The primary structure of the pbo-5 cDNA was determined by reverse transcription and polymerase chain reaction (RT-PCR). The pbo-5 cDNA includes an SL1 trans-spliced leader sequence at the 5’ end and a total of 9 exons spanning a 7 kb genomic region (Figure S4C). BLAST and protein motif queries of PBO-5 demonstrated that the predicted protein is a subunit of the ‘cys-loop’ ligand-gated ion channel superfamily. Each member of the ‘cys-loop’ protein family contains a signal peptide, an extracellular amino-terminus that contains consensus ligand-binding sites and an invariant disulfide bonded loop (the ‘cys-loop’), four transmembrane domains (M1-M4), a large cytoplasmic loop between M3-M4, and a short extracellular carboxy terminus (Karlin and Akabas, 1995; Ortells and Lunt, 1995) (Figure S5). Electron microscopy, electrophysiological and structural data suggest that ‘cys-loop’ ligand-gated ion channels are formed from five homologous subunits that are pseudo-symmetrically arranged around a central ion channel, such that the M2 domain lines the ion-channel wall and determines ion selectivity (Betz, 1990; Brejc et al., 2001; Unwin, 1993). Hydropathy plots and alignment of PBO-5 with various ligand-gated ion channel subunits demonstrates that PBO-5 contains the necessary features of a ‘cys-loop’ ligand-gated ion channel subunit (Figure S5).
Although many ‘cys-loop’ ligand-gated ion channel subunits have been identified, these subunits can usually be assigned to one of four neurotransmitter receptor families based on sequence similarity: the acetylcholine, serotonin, glycine or GABA families. Phylogenetic analysis demonstrates that PBO-5 represents a divergent subunit that cannot be categorized into one of the four families based on sequence similarity (Figure S6). Analysis of residues that determine ligand interactions did not identify a potential ligand for the PBO-5 receptor (Figure S5). Furthermore, mutants that are defective in GABA, acetylcholine, serotonin, and peptidergic transmission do not exhibit posterior body contraction defects. Therefore, it is unlikely that PBO-5 is activated by known classical neurotransmitters.
Because ‘cys-loop’ receptors are composed of homo- or heteropentamers, we scanned the genome to identify candidate proteins that may be required to form a functional receptor with PBO-5. Only one other gene, F11C7.1, which we call pbo-6, is closely related to pbo-5 (Figure S5 and S6). To determine the primary structure of PBO-6 we performed RT-PCR and isolated full-length cDNA clones. The full-length cDNA consists of 13 exons that span 4.1 kb of the genome. The PBO-6 cDNA encodes a 423 amino acid protein that shares 35% sequence identity with PBO-5 (Figure S5). The residues in the pore-forming M2 domains are identical with the exception of one conservative amino acid change. The C. elegans knockout consortium isolated a 1.9 kb deletion allele of pbo-6, removing residues 118−310, which encompasses the majority of the N-terminus and transmembrane domains M1-M3, and is therefore likely to be a null allele. Surprisingly, pbo-6(ok1564) animals exhibit normal posterior body contractions (66 pBocs/66 defecation cycles, n = 6), suggesting PBO-6 is not critical for the execution of the posterior body contraction in vivo.
If PBO-5 and PBO-6 are subunits of the receptor that mediates the posterior body contraction, then they should be expressed in the posterior body muscles. To determine the cellular expression of pbo-5, a transcriptional fusion gene was constructed that contained 3.8 kb upstream sequence of the translational start codon fused to the GFP open reading frame. To establish the expression pattern of PBO-6, we built a transcriptional fusion gene containing 4 kb of upstream promoter sequence fused to GFP. Animals carrying either the PBO-5 or PBO-6 transgene expressed GFP in the most-posterior body muscles (Figure 5), confirming these subunits are localized to the appropriate tissues to mediate posterior body muscle contraction.
To determine if PBO-5 and PBO-6 can assemble or co-assemble to form a functional receptor, we injected PBO-5 and PBO-6 cRNA alone or in combination into Xenopus oocytes. Agonists for other ligand-gated ion channels (ACh, GABA, glycine, 5-HT, glutamate, and choline) did not activate PBO-5, PBO-6 or PBO-5/6 injected eggs (data not shown). Failure of these neurotransmitters to activate the receptors was not unexpected as genetic evidence demonstrates that neither acetylcholine, GABA, glutamate or serotonin are required for the posterior body contraction.
We tested if H+ ions were capable of activating PBO-5, PBO-6 or PBO-5/6 injected oocytes. Test pulses of pH 6.0 did not elicit detectable currents from oocytes injected with PBO-6 cRNA alone (Figure 6A). Slight currents were detected when pH 6.0 test pulses were applied to PBO-5 injected oocytes, suggesting that PBO-5 can form a homomeric channel (−0.18 ± 0.06 μA; n = 18) (Figure 6A). This may explain the lack of an obvious phenotype in pbo-6 mutants. By contrast, PBO-5/6 injected eggs exhibited extremely robust currents when a pH 6.0 test pulse was applied (−3.9 ± 0.35 μA; n = 18) (Figure 6A). These data suggest that PBO-5/6 heteromultimerization is required for efficient functional receptor expression in vitro. To demonstrate current responses evoked by changes in pH were not due to endogenous channels or transporters, we applied maximal test pulses of pH 6.0 to naive oocytes or oocytes injected with water (n > 30). Only oocytes injected with PBO-5 or PBO-5/6 cRNA exhibited H+-gated responses, while uninjected cells exhibited no inward current in response to pH 6.0 test pulses.
To measure the pH50 (half-maximal activation) of putative PBO-5/6 heteromultimers, we first identified the pH range at which the recombinant receptors were activated. We determined that the pH10 (10% maximal activation) was approximately pH 7.0. The perfusion buffer was set at pH 7.4 for all experiments, where no activation of recombinant receptors was observed. A pH50 of 6.83 ± 0.01 was determined by applying decreasing pH test pulses (pH 7.2 − 6.0). A steep Hill coefficient of 5 suggests that PBO-5/6 receptors exhibit significant H+ ion binding cooperativity (Figure 6B and 6C).
H+ ions have been demonstrated to modulate classical synaptic transmission. For example, acidic changes inhibit acetylcholine receptor function, whereas alkaline environments enhance receptor function (Palma et al., 1991). Although none of the classical neurotransmitters tested could activate the PBO-5/6 receptor on their own, we sought to determine whether classical neurotransmitters co-activate PBO-5/6 receptors. Application of pH 6.8 solution plus 1mM acetylcholine, choline, GABA or glycine were not significantly different from pH 6.8 only application (Figure 6D). However, co-application of 5-HT did result in a small but significant increase in amplitude compared to controls (Figure 6D). Thus, the activity of the PBO receptor can be modulated by serotonin, although H+ ions alone are sufficient to activate the PBO receptor.
Acidification of the cleft could activate the posterior body contraction directly; alternatively, H+ ions may act as a secondary transmitter in alliance with another molecule. If H+ ions are sufficient to activate the posterior body contraction then application of exogenous H+ ions should be able to induce a posterior body contraction. We injected caged-protons into the coelomic space of wild-type worms. After recovery and mounting for observation, the animals received a brief flash of light (< 360 nm for 1−2 seconds) to uncage the protons. Injected animals exhibited a flash-induced posterior body contraction (45.4 ±8.8 μm, n=11) that was in phase with the flash stimulus (Figure 7A). Importantly, uninjected controls and FITC-dextran injected controls failed to activate a perceptible posterior body contraction in response to the light stimulus (Figure 7B). These results demonstrate that H+ ions can activate the posterior body contraction in vivo.
Although these data demonstrate that acidification can stimulate muscle contraction, they do not eliminate the possibility that pbo-4 provides some other essential component. To determine whether pbo-4 is specifically defective in proton secretion, we demonstrated that uncaging of H+ ions bypasses the requirement for PBO-4. We injected pbo-4 mutants with caged-protons and released them by flash photolysis. pbo-4 mutants normally do not initiate posterior body contractions (Figure 1B); however, when injected pbo-4 mutants were subjected to uncaging of H+ ions we observed posterior body contractions (52.8 ± 11.5 μm, n=7) that were in phase with the flash stimulus (Figure 7B). The flash-induced posterior body contractions appeared identical to those induced in the wild type and were not significantly different in mean contractile distance compared to wild type injected animals (t-test, P < 0.05). Finally, it was possible that H+ ions were causing a contraction through a toxic response rather than acting as a transmitter. However, uncaging of H+ ions in pbo-5(ox4) failed to elicit a posterior body contraction (Figure 7B). Thus, H+ ions are activating a specific receptor on the surface of the muscles, rather than causing a contraction by damaging cells.
Mutations in pbo-4 and pbo-5 specifically eliminate the posterior body contraction step of the defecation cycle motor program. Cycle timing, other contractions in the motor program, and body muscle function during locomotion are normal, suggesting that the mutations do not cause general defects in the health of the intestine or muscle, but rather are specifically involved in signaling between the intestine and the muscle. The simplest model is that protons released by the putative PBO-4 Na+/H+ exchanger activate the PBO receptor composed of PBO-5 and PBO-6; activation of this cation channel in turn depolarizes the cell and causes muscle contraction. This model is based on the following observations. First, the putative PBO-4 Na+/H+ exchanger is positioned to mediate communication with the muscles. PBO-4 is expressed in the posterior cells of the intestine, which act as the timekeeper for the defecation cycle (Dal Santo et al., 1999; Espelt et al., 2005), and the exchanger is localized to basolateral surface juxtaposed to the overlying body muscles. Second, PBO-4 mediates a transient acidification of the extracellular space preceding the posterior body contraction. Third, uncaging H+ ions in vivo is sufficient to activate normal posterior body contraction in wild-type or pbo-4 animals. Fourth, uncaged protons cannot bypass the pbo-5 defect, demonstrating that PBO-5 is a component of a specific proton receptor on the responding cell.
In 1958, Paton set out five criteria for admission of a molecule into the pantheon of neurotransmitters (Paton, 1958). Some of these rules have been set aside to accommodate gaseous and peptide transmitters (Baranano et al., 2001; Hyman, 2005); nevertheless, these rules provide a useful framework for considering whether a molecule acts as a neurotransmitter. An updated version (Cowan et al., 2001) of these rules include: (1) the chemical must be present in the signaling cell, (2) stimulation of the cell results in the release of the chemical, (3) there will be mechanisms for inactivating the transmitter, (4) the chemical must act on specific receptors on the responding cell, (5) exogenous application of the chemical must mimic the endogenous response, and (6) blocking the receptor blocks the activity of the neurotransmitter. In C. elegans, protons possess the attributes of a transmitter for muscle contraction. First, protons are found in all cells. Second, acidification of the extracellular space immediately precedes the posterior contraction. Third, although there is not a specific mechanism for clearing or depleting protons in the coelomic space, diffusion and biological buffers are likely to rapidly neutralize changes in pH. Fourth, protons act on a specific ligand-gated receptor, composed of the PBO-5 and PBO-6 proteins, which are localized to the posterior body muscles. Fifth, application of protons by releasing caged H+ ions stimulates the posterior body contraction. Sixth, genetic disruption of the receptor blocks the stimulation of muscle contraction by uncaged protons.
Neurotransmitters, of course, are released from neurons, whereas the H+ ion signal that we describe arises from the intestine. Thus, these experiments demonstrate that H+ ions are acting as a transmitter rather than a neurotransmitter. However, other Na+/H+ ion exchangers (Nehrke and Melvin, 2002) and the acid-sensitive receptor subunit PBO-5 are expressed in neurons. Thus, protons might act as a neurotransmitter at central synapses in C. elegans.
Consistent with a broader role for protons as intercellular messengers, a large family of proton-gated channels, termed the acid-sensing ion channels (ASICs), have been identified (Waldmann and Lazdunski, 1998). The ASICs are expressed in both the central and peripheral nervous system. For the most part these receptors are tuned to high acid environments and are noxious chemical receptors (Waldmann et al., 1997); however, studies indicate that some of these receptors may respond to physiological pH changes and act in intercellular signaling (Wemmie et al., 2002). Specifically, deletion of the mouse ASIC1 gene leads to deficits in learning and memory, suggesting H+ ion signaling is not confined to sensing noxious environments but is also important in central nervous system function (Wemmie et al., 2002). The source of H+ ions that activate the ASIC channels in the central nervous system is unknown. While repeated synaptic vesicle exocytosis may provide a limited supply of protons, other sources of “quick protons” such as Na+/H+ exchangers are attractive candidates for ASIC channel activation (Kaila and Ransom, 1998; Krishtal, 2003). Further studies may demonstrate that Na+/H+ ion exchangers function for the release of H+ ions acting as a neurotransmitter at central synapses in vertebrates.
Standard methods for maintaining C. elegans strains were used (Brenner, 1974). Bristol strain N2 was used as the wild type, and all animals were grown at room temperature (22.5°C).
pbo-4 alleles n2658 and sa300 were identified in EMS screens (E. M. Jorgensen and H.R. Horvitz, unpublished; J. Thomas, unpublished), ox10 was identified in a diepoxybutane screen, ok583 was identified in a UV/TMP screen (Knockout Consortium). Complementation tests confirmed that n2658, ox10 and sa300 are alleles of the same gene. n2658 was mapped to a chromosome using the mapping strains: EG1000 dpy-5(e61) I; rol-6(e187) II; lon-1(e1820) III and MT464 unc-5(e53) IV; dpy-119e224) V; lon-2(e678) X. Fine mapping on the X chromosome was performed using the following strains: EG1040 unc-2(ox2) dpy-6(e14) egl-15(n484), MT3001 dpy-6(e14) egl-15(n484), EM327 unc-115(e2225) vab-3(e648), and JT6885 unc-115(e2225) daf-12(m20) daf-11(m84).
pbo-5 alleles (n2331dm, ox7dm, ox4, ox5, ox9, n2303, n2330, sa242, sa243, and sa297) were identified in EMS (ethyl methanesulfonate) screens for defecation cycle defective mutants. Additional pbo-5 alleles (ox24, ox26, ox27, ox30, ox32, ox34, ox35, ox36, ox38, ox39) were identified in an EMS screen for suppressors of the dominant pbo-5(n2331sd) posterior body cramp. pbo-5 alleles were subsequently mapped to a chromosome using the mapping strains: EG1000 dpy-5(e61)I; rol-6(e187)II; lon-1(e1820)III and EG1020 bli-6(sc16)IV; dpy-11(e224)V; lon-2(e678)X. Fine mapping on chromosome V was done with the following strains: MT6931 egl-8(n2659) dpy-11(e224), MT2564 sqt-3(sc63) him-5(e1467) unc-76(e911), MT1505 dpy-21(e459) unc-51(e369), JK1520 fog-2(q71) pha-4(q496)/stu-3(q265) rol-9(sc148), JK1655 unc-51(e1189) pha-4(q506) rol-9(sc148)/fog-2(q71), SM3 fog-2(q71) rol-9(sc148) pha-4(q500)/stu-3(q265), MT6025 lin-31(n301) III; dpy-9(e12) IV; unc-51(e369) V, and MT6185 dpy-1(e1) III; unc-34(e566) V.
The defecation cycle of day 1 adult animals was scored for 11 cycles from at least 5 individual animals for cycle time and each step of the motor program. Error bars represent s.e.m.
Transgenic strains were generated by standard microinjection techniques (Mello et al., 1991). Rescue of pbo-4(n2658) mutants was obtained by co-injecting the cosmid K09C8 at 20ng/μl with 50 ng/μl pTG96, a plasmid that contains sur-5::GFP as a co-transformation marker. The pbo-4 genomic subclone (pPD58) was created by ligating an 8.5kb AgeI restriction fragment from cosmid K09C8 into pLitmus 28. pPD58 (20ng/μl) was co-injected with Pmyo-2:GFP (2ng/μl) and 1 kb plus DNA Ladder (Invitrogen) (78ng/μl) into pbo-4(ok583) animals. Stable lines (oxEx582) were obtained which were completely rescued for posterior body contraction.
Complementary cDNAs of the pbo-4 transcript were obtained by RT-PCR of wild type N2 RNA. The 5’ trans-spliced sequence was obtained by PCR amplification with an SL1 primer and a gene specific primer. The 3’ sequence was obtained by PCR amplification with an oligo-dT primer (Gibco) and a gene specific primer. Error free clones of these PCR products were identified to generate a full-length pbo-4 cDNA.
To identify the molecular lesions n2658, sa300 and ox10, we sequenced PCR products containing the K09C8.1 predicted open reading frame from worms of each genotype. The ok583 allele was isolated by the C. elegans Knockout Consortium. YACs, cosmids, plasmids and PCR products were prepared and injected into pbo-5 mutants by standard techniques. YAC deletions were generated by PCR-mediated gene disruption (Brachmann et al., 1998). DNA from mutant strains was prepared and amplified by single worm PCR to determine the nature of the molecular lesions. Deletion alleles were further characterized by PCR; point mutations were directly sequenced by an automated sequencing machine (Applied Biosystems).
The sequence of the pbo-5 transcript was determined by reverse transcription of wild-type mRNA followed by PCR amplification (RT-PCR) and direct sequencing. The 5’ trans-spliced sequence was obtained by PCR amplification using an SL1 primer and a nested, gene specific primer in the second exon. The 3’ sequence was obtained by PCR amplification with nested primers in the third predicted exons and with an oligo-dT primer. PCR products were cloned into the pCR2.1 TA cloning vector (Invitrogen) and a full-length error-free clone pPD68 was generated. For rescue of the pbo-5 mutant phenotype, a pbo-5 mini gene construct was made containing the Y44A6E.1 cDNA fused to 4.4 kb of sequences upstream of the second exon.
pbo-6 cDNAs were isolated by RT-PCR from polyA+ mRNA isolated from wild-type young adult hermaphrodites. We determined the 5’ end of the gene by circular RACE (Maruyama et al., 1995). The 3’ end of the gene was based on computer prediction and sequence similarity topbo-5. To isolate full-length cDNAs, oligonucleotide primers were designed to the 5’ and 3’ untranslated regions of pbo-6. PCR products were cloned into pCR2.1, and subsequent products were sequenced to generate a full-length error-free pbo-6 cDNA.
The full-length superecliptic pHlourin-GFP fusion (pAB16) was generated by ligating a 8.5 kb restriction fragment from pPD58 into pCR Blunt (Invitrogen). PCR primers with engineered ScaI restriction sites were used to PCR amplify the superecliptic GFP coding region from the vector pIA3 (Sankaranarayanan and Ryan, 2000). The 5.8 kb pbo-4 subclone was linearized with ScaI and de-phosphorylated, using Antarctic phosphatase (NEB). The superecliptic GFP open reading frame with ScaI ends was ligated into the linearized plasmid. pAB16 was injected into pbo-4(ok583) mutants at 20 ng/μl with Pmyo-2::GFP at 2 ng/μl and 1kb DNA Ladder (Invitrogen) at 78 ng/μl. Stable transgenic lines (EG3325 pbo-4(ok583); oxEx584 and EG3326 pbo-4(ok583); oxEx585) were obtained that exhibited GFP expression on the basolateral surface of the posterior intestine. These animals were also rescued for posterior body contraction. In order to image pHluorin in the pbo-5 mutant background, a stable transgenic line (EG4178 pbo-5(ox4); oxEx784) was generated by injecting pAB16 into pbo-5(ox4) mutants at 10 ng/μl with Pmyo-2::GFP at 1 ng/μl and lin-15(+) plasmid DNA at 90 ng/μl.
The vitellogenin promoter-driven superecliptic GFP fusion (pWD149) was generated by PCR of the pHluorin::GFP fusion from pAB16 using primers that introduce NcoI and SphI sites flanking the gene. This PCR fragment was cloned into a plasmid pGEM-Pvit-2 (courtesy of Maureen Peters) carrying the promoter for the vitellogenin gene, vit-2. pWD149 was injected into pbo-4(n2658) mutants at 10 ng/μl with Pmyo-2::GFP at 1 ng/μl and lin-15(+) plasmid DNA at 90 ng/μl. Stable transgenic lines (EG4077 pbo-4(n2658); oxEx770) were obtained that exhibited dim GFP expression in all intestinal cells of one day old adults and bright expression in older animals. Young adults exhibited rescue; old animals with high PBO-4 expression exhibited defects in the posterior body contraction, perhaps due to desensitization of PBO-5.
To image pHluorin in a pbo-4 mutant background, a non-rescuing version of pAB16 was generated by deleting the cytoplasmic C-terminus of PBO-4 after amino acid 509 (immediately following transmembrane 12). pAB16 was digested with PmeI and BstZ17I and religated to produce pWD153. A stable transgenic line was created (EG4176 pbo-4(n2658); oxEx782) by injecting pbo-4(n2658) mutants with pWD153 at 10 ng/μl, Pmyo-2::GFP at 1 ng/μl and lin-15(+) plasmid DNA at 90 ng/μl.
The pbo-5 transcriptional reporter construct contains a 3.8 kb PCR-amplified fragment upstream of the translational start codon fused to the GFP open reading frame, followed by a stop codon and the polyadenylation site from the unc-54 gene (pPD54). The pbo-6 transcriptional reporter construct contains a 4.0 kb PCR-amplified promoter region fused to the GFP open reading frame followed by a stop codon and the polyadenylation site from the unc-54 gene (pPD72). These GFP constructs were injected into lin-15(n765ts) animals at 10ng/μl along with lin-15(+) (pL15EK) as a co-transformation marker at 30ng/μl (Clark et al., 1994) and 1 kb+ DNA ladder at 70ng/μl. Stable transgenic lines were obtained that expressed GFP in the posterior body wall muscles for pbo-5::gfp (EG2599 lin-15(n765ts); oxEx355) and for pbo-6::gfp (EG3322 lin-15(n765ts); oxEx581).
Young adult animals were immobilized with cyanoacrylate glue on an agarose pad formed on a standard microscope slide. Animals were glued along the length of one side of the body with an effort to leave the very posterior end free to contract. To promote pharyngeal pumping, a necessary activity needed for posterior body contractions, animals were rinsed with 10mM serotonin (Sigma), covered with a cover glass, and the edges were sealed with Vaseline. This preparation enabled us to cleanly visualize acid transients produced by pHluorin::PBO-4 quenching with a high numerical aperture objective [63×, 1.4 N.A. plan apochromat objective (Zeiss)] in wide-field on an inverted microscope (iMIC, TILL Photonics). While serotonin increased the likelihood of capturing a pBoc event in rescued animals during the recording period, serotonin is known to produce irregularities in the periodicity of the cycle (E.M.J, unpublished observations). This combined with the glued restraint of the animals under a coverslip, we did not expect to see regular periodic cycles. However, we did observe three of eighteen six-minute recordings with astonishing ~50 s periodic events. Animals expressing PBO-4::pHluorin were excited with a 488 ± 15nm light from a monochrometer (Polychrome V, TILL Photonics) with a 50ms exposure time at 10 Hz. Individuals were typically recorded over a period of six minutes. Images were recorded with 4×4 binning on a 14-bit cooled-CCD camera (Andor Technology).
Regions of interest were defined as an area slightly larger than the posterior intestine cells expressing PBO-4::pHluorin in the first frame of the image sequence. Mean pixel intensity of the ROI was computed in each frame of the image sequence using TILLvisION (TILL Photonics). Over the six minute acquisition period, an exponential decrement due to photobleaching was observed (~20% of the initial fluorescence was lost at the end of a six minute session). To measure relative amplitudes of fluorescence changes, the effect of photobleaching was removed by normalizing the baseline to the exponential decline using Igor Pro (Wavemetrics). Corrected traces were further analyzed to measure event amplitude, rise time, and decay using an event detection program (MiniAnalysis, Synaptosoft). Statistical tests were performed using InStat (GraphPad). To identify characteristic posterior body contractions image sequences were manually inspected. If motion of any kind was observed, the frame in which the motion initiated was noted and compared to the quantitative time course. pHluorin (non-motion induced) events were downwardly directed with a fast rise time and slow exponential decay. Spurious motion of the tail could be seen in quantitative time courses as events that were both positively and negatively directed with irregular kinetics.
To generate plasmid constructs for Xenopus oocyte expression, we subcloned full length error-free cDNAs into the pSGEM expression vector (Villmann et al., 1997). The pbo-5 expression vector was constructed by cutting the pPD70 plasmid with restriction enzymes SacII and EagI, then using T4 DNA polymerase to blunt the ends. Cut products were gel purified (Qiagen) and re-ligated using T4 DNA ligase (Promega) to create the plasmid pAB20. The PBO-6 expression construct was generated from a previously sequenced error-free cDNA (M. Peter, unpublished). A blunt-end cDNA fragment was cloned into the HincII site of the pSGEM expression vector to produce pAB21.
Capped RNA was prepared using the T7 mMessage mMachine kit (Ambion). Xenopus oocytes were collected and injected with 25 ng each of PBO-5 and/or PBO-6 cRNA and two-electrode voltage clamp recordings were performed 3−5 days post-injection. For most experiments the standard bath solution for dose-response and control I-V experiments was Ringer's (in mM): 115 NaCl, 1.8 BaCl2, 10 Bis-Tris Propane (pH 7.4 Acetic acid). For dose-response experiments, each oocyte was subjected to a 5 second application of test pH (7.0 − 5.0) with 2 minutes of pH 7.4 wash between test applications. Buffers used were either Bis-Tris Propane (1,3-Bis[tris(hydroxymethyl)methylamino]propane), MES (2-(N-Morpholino)-ethanesulfonic acid) or HEPES (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid). Before each experiment, at least two control oocytes were voltage clamped at −60mV and maximal pH 6.0 test pulses were applied to ensure that individual batches of oocytes did not express endogenous proton-activated channels that could obscure analysis.
Data acquisition and analysis were performed using Axograph (Axon Instruments) software, and curve fitting and statistical analysis were performed with Prism (Graphpad). Dose-response curves from individual oocytes were normalized to the maximum and minimum values and averaged for at least eleven oocytes. Normalized data were fit to the four-parameter equation derived from the Hill equation: Y=Min + (Max-Min)/(1 + 10(LogEC50-X)(nH)), where Max is the maximal response, Min is the response at the lowest drug concentration, X is the logarithm of agonist concentration, EC50 is the half-maximal response, and nH is the Hill coefficient. Error bars represent the standard error of the mean.
Adult animals were injected with 0.1 mg/ml NPE-caged proton (2-hydroxyphenyl-1-(2-nitrophenyl)ethyl phosphate, sodium salt, and 0.1 mg/ml FITC-Dextran (fluorescein-dextran, sodium salt, 10,000 MW) as a co-injection marker. The caged-H+ ion and FITC-Dextran were dissolved in 5mM Na+-phosphate (pH 7.0). Animals were mounted on an agarose pad and injected in the coelomic space. Injections were done adjacent to the vulva to avoid damaging the posterior body muscles with the needle. Successful injections were determined by scoring for the co-injection marker FITC-dextran; these animals were allowed to recover for 25−30 minutes in the dark on nematode growth plates. Animals were observed for 1−2 defecation cycles to determine if the animal was behaving normally. Next, we either mounted the animal on a cooled 2% agarose pad covered with bacteria, or kept the animals on agarose plates with bacteria. A modified DAPI filter set that allowed < 360 nm and a manual shutter were used to apply UV light to the injected animal for 1−3 seconds. Digital video recordings were acquired directly to a computer through a cooled CCD camera.
Quantification was performed offline by reviewing recorded video using iMovie (Apple). Movies were played in slow motion and still frames were saved for individual animals immediately preceding the flash, immediately after the flash, and after recovery. To quantify the mean contractile distance, we measured from the vulva to the posterior tip of the tail for each saved frame. We used the vulva as a landmark that would be consistent from animal to animal. Distances were measured using ImageJ (NIH). At least 5 animals were quantified for each genotype.
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