Cell excitability necessary for male mating behavior in Caenorhabditis elegans is coordinated by interactions between big current and ether-a-go-go family K(+) channels

Variations in K(+) channel composition allow for differences in cell excitability and, at an organismal level, provide flexibility to behavioral regulation. When the function of a K(+) channel is disrupted, the remaining K(+) channels might incompletely compensate, manifesting as abnormal organismal behavior. In this study, we explored how different K(+) channels interact to regulate the neuromuscular circuitry used by Caenorhabditis elegans males to protract their copulatory spicules from their tail and insert them into the hermaphrodite's vulva during mating. We determined that the big current K(+) channel (BK)/SLO-1 genetically interacts with ether-a-go-go (EAG)/EGL-2 and EAG-related gene/UNC-103 K(+) channels to control spicule protraction. Through rescue experiments, we show that specific slo-1 isoforms affect spicule protraction. Gene expression studies show that slo-1 and egl-2 expression can be upregulated in a calcium/calmodulin-dependent protein kinase II-dependent manner to compensate for the loss of unc-103 and conversely, unc-103 can partially compensate for the loss of SLO-1 function. In conclusion, an interaction between BK and EAG family K(+) channels produces the muscle excitability levels that regulate the timing of spicule protraction and the success of male mating behavior.     

The CaMKII UNC-43 activates the MAPKKK NSY-1 to execute a lateral signaling decision required for asymmetric olfactory neuron fates

A stochastic cell fate decision mediated by axon contact and calcium signaling causes one of the two bilaterally symmetric AWC neurons, either AWCL or AWCR, to express the candidate olfactory receptor str-2. nsy-1 mutants express str-2 in both neurons, disrupting AWC asymmetry. nsy-1 encodes a homolog of the human MAP kinase kinase kinase (MAPKKK) ASK1, an activator of JNK and p38 kinases. Based on genetic epistasis analysis, nsy-1 appears to act downstream of the CaMKII unc-43, and NSY-1 associates with UNC-43, suggesting that UNC-43/CaMKII activates the NSY-1 MAP kinase cassette. Mosaic analysis demonstrates that UNC-43 and NSY-1 act primarily in a cell-autonomous execution step that represses str-2 expression in one AWC cell, downstream of the initial lateral signaling pathway that coordinates the fates of the two cells.     

An Alpha-Catulin Homologue Controls Neuromuscular Function through Localization of the Dystrophin Complex and BK Channels in Caenorhabditis elegans

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.     

C. elegans Demonstrates Distinct Behaviors within a Fixed and Uniform Electric Field

C. elegans will orient and travel in a straight uninterrupted path directly towards the negative pole of a DC electric field. We have sought to understand the strategy worms use to navigate to the negative pole in a uniform electric field that is fixed in both direction and magnitude. We examined this behavior by quantifying three aspects of electrotaxis behavior in response to different applied field strengths: the mean approach trajectory angles of the animals’ tracks, turning behavior (pirouettes) and average population speeds. We determined that C. elegans align directly to the negative pole of an electric field at sub-preferred field strength and alter approach trajectories at higher field strengths to maintain taxis within a preferred range we have calculated to be ~ 5V/cm. We sought to identify the sensory neurons responsible for the animals’ tracking to a preferred field strength. eat-4 mutant animals defective in glutamatergic signaling of the amphid sensory neurons are severely electrotaxis defective and ceh-36 mutant animals, which are defective in the terminal differentiation of two types of sensory neurons, AWC and ASE, are partially defective in electrotaxis. To further elucidate the role of the AWC neurons, we examined the role of each of the pair of AWC neurons (AWC^OFF and AWC^ON), which are functionally asymmetric and express different genes. nsy-5/inx-19 mutant animals, which express both neurons as AWC^OFF, are severely impaired in electrotaxis behavior while nsy-1 mutants, which express both neurons as AWC^ON, are able to differentiate field strengths required for navigation to a specific field strength within an electric field. We also tested a strain with targeted genetic ablation of AWC neurons and found that these animals showed only slight disruption of directionality and turning behavior. These results suggest a role for AWC neurons in which complete loss of function is less disruptive than loss of functional asymmetry in electrotaxis behavior within a uniform fixed field.     

SEK-1 MAPKK mediates Ca2+ signaling to determine neuronal asymmetric development in Caenorhabditis?elegans

The mitogen-activated protein kinase (MAPK) pathway is a highly conserved signaling cascade that converts extracellular signals into various outputs. In Caenorhabditis elegans, asymmetric expression of the candidate odorant receptor STR-2 in either the left or the right of two bilaterally symmetrical olfactory AWC neurons is regulated by axon contact and Ca²⁺ signaling. We show that the MAPK kinase (MAPKK) SEK-1 is required for asymmetric expression in AWC neurons. Genetic and biochemical analyses reveal that SEK-1 functions in a pathway downstream of UNC-43 and NSY-1, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and MAPK kinase kinase (MAPKKK), respectively. Thus, the NSY-1–SEK-1–MAPK cascade is activated by Ca²⁺ signaling through CaMKII and establishes asymmetric cell fate decision during neuronal development.     

Microtubule-based localization of a synaptic calcium-signaling complex is required for left-right neuronal asymmetry in C. elegans

The axons of C. elegans left and right AWC olfactory neurons communicate at synapses through a calcium-signaling complex to regulate stochastic asymmetric cell identities called AWC(ON) and AWC(OFF). However, it is not known how the calcium-signaling complex, which consists of UNC-43/CaMKII, TIR-1/SARM adaptor protein and NSY-1/ASK1 MAPKKK, is localized to postsynaptic sites in the AWC axons for this lateral interaction. Here, we show that microtubule-based localization of the TIR-1 signaling complex to the synapses regulates AWC asymmetry. Similar to unc-43, tir-1 and nsy-1 loss-of-function mutants, specific disruption of microtubules in AWC by nocodazole generates two AWC(ON) neurons. Reduced localization of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons strongly correlates with the 2AWC(ON) phenotype in nocodazole-treated animals. We identified kinesin motor unc-104/kif1a mutants for enhancement of the 2AWC(ON) phenotype of a hypomorphic tir-1 mutant. Mutations in unc-104, like microtubule depolymerization, lead to a reduced level of UNC-43, TIR-1 and NSY-1 proteins in the AWC axons. In addition, dynamic transport of TIR-1 in the AWC axons is dependent on unc-104, the primary motor required for the transport of presynaptic vesicles. Furthermore, unc-104 acts non-cell autonomously in the AWC(ON) neuron to regulate the AWC(OFF) identity. Together, these results suggest a model in which UNC-104 may transport some unknown presynaptic factor(s) in the future AWC(ON) cell that non-cell autonomously control the trafficking of the TIR-1 signaling complex to postsynaptic regions of the AWC axons to regulate the AWC(OFF) identity.     

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann^1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor³ for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L''Etoile et al.,⁴ uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC⁴. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC⁵. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC^6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.     

Regulators of AWC-Mediated Olfactory Plasticity in Caenorhabditis elegans

While most sensory neurons will adapt to prolonged stimulation by down-regulating their responsiveness to the signal, it is not clear which events initiate long-lasting sensory adaptation. Likewise, we are just beginning to understand how the physiology of the adapted cell is altered. Caenorhabditis elegans is inherently attracted to specific odors that are sensed by the paired AWC olfactory sensory neurons. The attraction diminishes if the animal experiences these odors for a prolonged period of time in the absence of food. The AWC neuron responds acutely to odor-exposure by closing calcium channels. While odortaxis requires a Gα subunit protein, cGMP-gated channels, and guanylyl cyclases, adaptation to prolonged odor exposure requires nuclear entry of the cGMP-dependent protein kinase, EGL-4. We asked which candidate members of the olfactory signal transduction pathway promote nuclear entry of EGL-4 and which molecules might induce long-term adaptation downstream of EGL-4 nuclear entry. We found that initiation of long-term adaptation, as assessed by nuclear entry of EGL-4, is dependent on G-protein mediated signaling but is independent of fluxes in calcium levels. We show that long-term adaptation requires polyunsaturated fatty acids (PUFAs) that may act on the transient receptor potential (TRP) channel type V OSM-9 downstream of EGL-4 nuclear entry. We also present evidence that high diacylglycerol (DAG) levels block long-term adaptation without affecting EGL-4 nuclear entry. Our analysis provides a model for the process of long-term adaptation that occurs within the AWC neuron of C. elegans: G-protein signaling initiates long-lasting olfactory adaptation by promoting the nuclear entry of EGL-4, and once EGL-4 has entered the nucleus, processes such as PUFA activation of the TRP channel OSM-9 may dampen the output of the AWC neuron.     

SLO-1-channels of parasitic nematodes reconstitute locomotor behaviour and emodepside sensitivity in Caenorhabditis elegans slo-1 loss of function mutants

The calcium-gated potassium channel SLO-1 in Caenorhabditis elegans was recently identified as key component for action of emodepside, a new anthelmintic drug with broad spectrum activity. In this study we identified orthologues of slo-1 in Ancylostoma caninum, Cooperia oncophora, and Haemonchus contortus, all important parasitic nematodes in veterinary medicine. Furthermore, functional analyses of these slo-1 orthologues were performed using heterologous expression in C. elegans. We expressed A. caninum and C. oncophora slo-1 in the emodepside-resistant genetic background of the slo-1 loss-of-function mutant NM1968 slo-1(js379). Transformants expressing A. caninum slo-1 from C. elegans slo-1 promoter were highly susceptible (compared to the fully emodepside-resistant slo-1(js379)) and showed no significant difference in their emodepside susceptibility compared to wild-type C. elegans (p = 0.831). Therefore, the SLO-1 channels of A. caninum and C. elegans appear to be completely functionally interchangeable in terms of emodepside sensitivity. Furthermore, we tested the ability of the 5′ flanking regions of A. caninum and C. oncophora slo-1 to drive expression of SLO-1 in C. elegans and confirmed functionality of the putative promoters in this heterologous system. For all transgenic lines tested, expression of either native C. elegans slo-1 or the parasite-derived orthologue rescued emodepside sensitivity in slo-1(js379) and the locomotor phenotype of increased reversal frequency confirming the reconstitution of SLO-1 function in the locomotor circuits. A potent mammalian SLO-1 channel inhibitor, penitrem A, showed emodepside antagonising effects in A. caninum and C. elegans. The study combined the investigation of new anthelmintic targets from parasitic nematodes and experimental use of the respective target genes in C. elegans, therefore closing the gap between research approaches using model nematodes and those using target organisms. Considering the still scarcely advanced techniques for genetic engineering of parasitic nematodes, the presented method provides an excellent opportunity for examining the pharmacofunction of anthelmintic targets derived from parasitic nematodes.     

An innexin-dependent cell network establishes left-right neuronal asymmetry in C. elegans

Gap junctions are widespread in immature neuronal circuits, but their functional significance is poorly understood. We show here that a transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of Caenorhabditis elegans. nsy-5 is required for the left and right AWC olfactory neurons to establish stochastic, asymmetric patterns of gene expression during embryogenesis. nsy-5-dependent gap junctions in the embryo transiently connect the AWC cell bodies with those of numerous other neurons. Both AWCs and several other classes of nsy-5-expressing neurons participate in signaling that coordinates left-right AWC asymmetry. The right AWC can respond to nsy-5 directly, but the left AWC requires nsy-5 function in multiple cells of the network. NSY-5 forms hemichannels and intercellular gap-junction channels in Xenopus oocytes, consistent with a combination of cell-intrinsic and network functions. These results provide insight into gap-junction activity in developing circuits.     

Worms take to the slo lane: a perspective on the mode of action of emodepside

The cyclo-octapdepsipeptide anthelmintic emodepside exerts a profound paralysis on parasitic and free-living nematodes. The neuromuscular junction is a significant determinant of this effect. Pharmacological and electrophysiological analyses in the parasitic nematode Ascaris suum have resolved that emodepside elicits a hyperpolarisation of body wall muscle, which is dependent on extracellular calcium and the efflux of potassium ions. The molecular basis for emodepside's action has been investigated in forward genetic screens in the free-living nematode Caenorhabditis elegans. Two screens for emodepside resistance, totalling 20,000 genomes, identified several mutants of slo-1, which encodes a calcium-activated potassium channel homologous to mammalian BK channels. Slo-1 null mutants are more than 1000-fold less sensitive to emodepside than wild-type C. elegans and tissue-specific expression studies show emodepside acts on SLO-1 in neurons regulating feeding and motility as well as acting on SLO-1 in body wall muscle. These genetic data, combined with physiological measurements in C. elegans and the earlier physiological analyses on A. suum, define a pivotal role for SLO-1 in the mode of action of emodepside. Additional signalling pathways have emerged as determinants of emodepside's mode of action through biochemical and hypothesis-driven approaches. Mutant analyses of these pathways suggest a modulatory role for each of them in emodepside's mode of action; however, they impart much more modest changes in the sensitivity to emodepside than mutations in slo-1. Taken together these studies identify SLO-1 as the major determinant of emodepside's anthelmintic activity. Structural information on the BK channels has advanced significantly in the last 2?years. Therefore, we rationalise this possibility by suggesting a model that speculates on the nature of the emodepside pharmacophore within the calcium-activated potassium channels.     

The claudin superfamily protein nsy-4 biases lateral signaling to generate left-right asymmetry in C. elegans olfactory neurons

Early in C. elegans development, signaling between bilaterally symmetric AWC olfactory neurons causes them to express different odorant receptor genes. AWC left-right asymmetry is stochastic: in each animal, either the left or the right neuron randomly becomes AWC(ON), and the other neuron becomes AWC(OFF). Here we show that the nsy-4 gene coordinates the lateral signaling that diversifies AWC(ON) and AWC(OFF) neurons. nsy-4 mutants generate 2 AWC(OFF) neurons, as expected if communication between the AWC neurons is lost, whereas overexpression of nsy-4 results in 2 AWC(ON) neurons. nsy-4 encodes a transmembrane protein related to the gamma subunits of voltage-activated calcium channels and the claudin superfamily; it interacts genetically with calcium channels and antagonizes a calcium-to-MAP kinase cascade in the neuron that becomes AWC(ON). Genetic mosaic analysis indicates that nsy-4 functions both cell-autonomously and nonautonomously in signaling between AWC neurons, providing evidence for lateral signaling and feedback that coordinate asymmetric receptor choice.     

The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.     

SLO-2 isoforms with unique Ca2+- and voltage-dependence characteristics confer sensitivity to hypoxia in C. elegans

Slo channels are large conductance K⁺ channels that display marked differences in their gating by intracellular ions. Among them, the Slo1 and C. elegans SLO-2 channels are gated by calcium (Ca²⁺), while mammalian Slo2 channels are activated by both sodium (Na⁺) and chloride (Cl⁻). Here, we report that SLO-2 channels, SLO-2a and a novel N-terminal variant isoform, SLO-2b, are activated by Ca²⁺ and voltage, but in contrast to previous reports they do not exhibit Cl⁻ sensitivity. Most importantly, SLO-2 provides a unique case in the Slo family for sensing Ca²⁺ with the high-affinity Ca²⁺ regulatory site in the RCK1 but not the RCK2 domain, formed through interactions with residues E319 and E487 (that correspond to D362 and E535 of Slo1, respectively). The SLO-2 RCK2 domain lacks the Ca²⁺ bowl structure and shows minimal Ca²⁺ dependence. In addition, in contrast to SLO-1, SLO-2 loss-of-function mutants confer resistance to hypoxia in C. elegans. Thus, the C. elegans SLO-2 channels possess unique biophysical and functional properties.     

The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans

The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWC(ON) and AWC(OFF), by inhibiting a calcium-mediated signaling pathway in the future AWC(ON) cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWC(ON) fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWC(ON) cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWC(ON) identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWC(ON) neurons. tir-1 expression is downregulated through its 3' UTR in AWC(ON), in which mir-71 is expressed at a higher level than in AWC(OFF). In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3' UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWC(ON) identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.     

SLO,SLO, quick,quick, slow: calcium-activated potassium channels as regulators of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> behaviour and targets for anthelmintics

Large-conductance calcium and voltage-activated potassium channels, termed SLO-1 (or BK), are pivotal players in the regulation of cell excitability across the animal phyla. Furthermore, emerging evidence indicates that these channels are key mediators of a number of neuroactive drugs, including the most recent new anthelmintic, the cyclo-octadepsipeptide emodepside. Detailed reviews of the structure, function and pharmacology of BK channels have recently been provided (Salkoff et al. in Nat Rev Neurosci 7:921–931, 2006; Ghatta et al. in Pharmacol Ther 110:103–116, 2006) and therefore these aspects will only briefly be covered here. The purpose of this review is to discuss how SLO-1 channels might function as regulators of neural transmission and network activity. In particular, we focus on the role of SLO-1 in the regulation of Caenorhabditis elegans behaviour and highlight the role of this channel as an effector for pleiotropic actions of neuroactive drugs, including emodepside. On the premise that C. elegans is a ‘model nematode’ with respect to many aspects of neural function, the intention is that this might inform a broader understanding of the role of these channels in the nematodes and their potential as novel anthelmintic targets.     

Putative calcium‐binding domains of the Caenorhabditis elegans BK channel are dispensable for intoxication and ethanol activation

Alcohol modulates the highly conserved, voltage‐ and calcium‐activated potassium (BK) channel, which contributes to alcohol‐mediated behaviors in species from worms to humans. Previous studies have shown that the calcium‐sensitive domains, RCK1 and the Ca²⁺ bowl, are required for ethanol activation of the mammalian BK channel in vitro. In the nematode Caenorhabditis elegans, ethanol activates the BK channel in vivo, and deletion of the worm BK channel, SLO‐1, confers strong resistance to intoxication. To determine if the conserved RCK1 and calcium bowl domains were also critical for intoxication and basal BK channel‐dependent behaviors in C. elegans, we generated transgenic worms that express mutated SLO‐1 channels predicted to have the RCK1, Ca²⁺ bowl or both domains rendered insensitive to calcium. As expected, mutating these domains inhibited basal function of SLO‐1 in vivo as neck and body curvature of these mutants mimicked that of the BK null mutant. Unexpectedly, however, mutating these domains singly or together in SLO‐1 had no effect on intoxication in C. elegans. Consistent with these behavioral results, we found that ethanol activated the SLO‐1 channel in vitro with or without these domains. By contrast, in agreement with previous in vitro findings, C. elegans harboring a human BK channel with mutated calcium‐sensing domains displayed resistance to intoxication. Thus, for the worm SLO‐1 channel, the putative calcium‐sensitive domains are critical for basal in vivo function but unnecessary for in vivo ethanol action.     

Caenorhabditis elegans junctophilin has tissue-specific functions and regulates neurotransmission with extended-synaptotagmin

The junctophilin family of proteins tether together plasma membrane (PM) and endoplasmic reticulum (ER) membranes, and couple PM- and ER-localized calcium channels. Understanding in vivo functions of junctophilins is of great interest for dissecting the physiological roles of ER-PM contact sites. Here, we show that the sole Caenorhabditis elegans junctophilin JPH-1 localizes to discrete membrane contact sites in neurons and muscles and has important tissue-specific functions. jph-1 null mutants display slow growth and development due to weaker contraction of pharyngeal muscles, leading to reduced feeding. In the body wall muscle, JPH-1 colocalizes with the PM-localized EGL-19 voltage-gated calcium channel and ER-localized UNC-68 RyR calcium channel, and is required for animal movement. In neurons, JPH-1 colocalizes with the membrane contact site protein Extended-SYnaptoTagmin 2 (ESYT-2) in the soma, and is present near presynaptic release sites. Interestingly, jph-1 and esyt-2 null mutants display mutual suppression in their response to aldicarb, suggesting that JPH-1 and ESYT-2 have antagonistic roles in neuromuscular synaptic transmission. Additionally, we find an unexpected cell nonautonomous effect of jph-1 in axon regrowth after injury. Genetic double mutant analysis suggests that jph-1 functions in overlapping pathways with two PM-localized voltage-gated calcium channels, egl-19 and unc-2, and with unc-68 for animal health and development. Finally, we show that jph-1 regulates the colocalization of EGL-19 and UNC-68 and that unc-68 is required for JPH-1 localization to ER-PM puncta. Our data demonstrate important roles for junctophilin in cellular physiology, and also provide insights into how junctophilin functions together with other calcium channels in vivo.     

Noncell- and cell-autonomous G-protein-signaling converges with Ca2+/mitogen-activated protein kinase signaling to regulate str-2 receptor gene expression in Caenorhabditis elegans

In the sensory system of C. elegans, the candidate odorant receptor gene str-2 is strongly expressed in one of the two AWC neurons and weakly in both ASI neurons. Asymmetric AWC expression results from suppression of str-2 expression by a Ca2+/MAPK signaling pathway in one of the AWC neurons early in development. Here we show that the same Ca2+/MAPK pathway promotes str-2 expression in the AWC and ASI neurons together with multiple cell-autonomous and noncell-autonomous G-protein-signaling pathways. In first-stage larvae and adult animals, signals mediated by the Galpha subunits ODR-3, GPA-2, GPA-5, and GPA-6 and a Ca2+/MAPK pathway involving the Ca2+ channel subunit UNC-36, the CaMKII UNC-43, and the MAPKK kinase NSY-1 induce strong str-2 expression. Cell-specific rescue experiments suggest that ODR-3 and the Ca2+/MAPK genes function in the AWC neurons, but that GPA-5 and GPA-6 function in the AWA and ADL neurons, respectively. In Dauer larvae, the same network of genes promotes strong str-2 expression in the ASI neurons, but ODR-3 functions in AWB and ASH and GPA-6 in AWB. Our results reveal a complex signaling network, encompassing signals from multiple cells, that controls the level of receptor gene expression at different developmental stages.