The C. elegans cGMP-dependent protein kinase EGL-4 regulates nociceptive behavioral sensitivity

Abstract

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.

Figure 1. C. elegans EGL-4 regulates quinine sensitivity in ASH.

(A) egl-4(lof) animals respond better than wild-type animals to dilute (1 mM) quinine, while egl-4(gof) animals show a decreased sensitivity to 10 mM quinine, when compared to wild-type animals. p<0.0001 for each. (B) The ASH sensory neurons are the primary neurons used to detect quinine, but the ASK neurons also contribute . The osm-10 , srb-6 and srbc-66 promoters were used to drive expression of wild-type egl-4 in egl-4(lof) animals. The osm-10 promoter expresses in ASH, ASI, PHA and PHB, while the srb-6 promoter drives expression in ASH, ADL, ADF, PHA and PHB. ASH is the only head sensory neuron common to both promoters. The srbc-66 promoter expresses in ASK. While egl-4(lof) animals respond better than wild-type animals to 1 mM quinine, restoring EGL-4 function in ASH significantly diminished this hypersensitivity (p<0.0001 for both). EGL-4 expression in ASK had no effect (p>0.5). (C) RNAi knock-down of egl-4 in the ASH sensory neurons of otherwise wild-type animals, using the osm-10 or srb-6 promoter resulted in behavioral hypersensitivity to dilute (1 mM) quinine, similar to egl-4(lof) animals (p<0.0001 when compared to N2 animals for both transgenes). The percentage of animals responding is shown. The combined data of ≥3 independent lines, n≥120 transgenic animals, is shown. Error bars represent the standard error of the mean (SEM). Alleles used: egl-4(n479) loss-of-function and egl-4(ad450) gain-of-function. WT = the N2 wild-type strain. lof = loss-of-function. gof = gain-of-function.

Figure 2. EGL-4 does not regulate ASH sensitivity in general.

C. elegans respond to bitter stimuli in addition to quinine. (A) Animals lacking EGL-4 function are hypersensitive to dilute amodiaquine (p≤0.01 when compared to wild-type animals), but not dilute primaquine (B) (p≥0.05). The percentage of animals responding is shown. The ASH sensory neurons also detect the volatile odorant octanol, the heavy metal copper and the detergent SDS. (C) egl-4(lof) mutant animals are moderately hypersensitive to dilute octanol (p<0.02). Time to respond is shown. (D–E) egl-4(lof) animals respond similarly to wild-type animals to both copper and SDS, across a range of concentrations (p>0.1 for each concentration, except p = 0.03 for 1 mM copper). The percentage of animals responding is shown. n>40 for each. All tastants were dissolved in M13 buffer, pH 7.4. Error bars represent the standard error of the mean (SEM). Allele used: egl-4(n479) loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function.

Figure 3. EGL-4 functions in the cytoplasm to regulate calcium signaling.

(A) EGL-4 functions in the cytoplasm to regulate behavioral sensitivity to quinine. The osm-10 promoter was used to express wild-type EGL-4, EGL-4 lacking its endogenous nuclear localization sequence (NLS) or EGL-4 with an additional NLS in the ASH sensory neurons of egl-4(lof) animals. In each case, the EGL-4 was expressed as a fusion with the green fluorescent protein (GFP). Wild-type GFP−EGL-4 was localized throughout the cell and rescued the quinine hypersensitivity of egl-4(lof) animals. GFP−EGL-4(ΔNLS) was restricted to the cytoplasm and rescued the hypersensitivity of egl-4(lof) animals as well as wild-type GFP−EGL-4 (p>0.05). NLS−GFP−EGL-4 was sequestered to the nucleus and had only a small, but statistically significant, rescuing effect (p<0.001). (B) Stimulus-evoked calcium transients in the ASH neurons are enhanced in egl-4(lof) animals. The sra-6 promoter was used to express the genetically encoded calcium indicator G-CaMP3 in the ASH sensory neurons, kyEx2865 (sra-6p::G-CaMP3;ofm-1p::gfp) . Using a microfluidic device, an adult animal was restrained while quinine was delivered to its nose for 10 seconds (black horizontal bar), and the change in fluorescence intensity was recorded. The average ratio change ± the standard error of the mean (SEM) is indicated on each trace. n = 10 animals for each condition. (C) The averaged maximum evoked calcium change for 10, 1 and 0 mM quinine is shown. egl-4(lof) animals showed an elevated ASH calcium flux upon exposure to 1 mM quinine when compared to wild-type animals (p<0.05) that is rescued by osm-10p::egl-4 expression in ASH (p>0.1 when compared to wild-type animals). Error bars represent the SEM. Alleles used: egl-4(n479) loss-of-function and egl-4(ad450) gain-of-function. WT = the N2 wild-type strain. lof = loss-of-function, gof = gain-of-function. s = seconds.

Figure 4. RGS proteins are targets of EGL-4.

(A) Animals lacking each of the 8 neuronally expressed RGS proteins were tested for response to 1 mM quinine. rgs-2(lof) and rgs-3(lof) animals respond better than wild-type animals to dilute (1 mM) quinine (p<0.001). (B) RNAi knock-down of rgs-2 or rgs-3 in the quinine-detecting ASH sensory neurons of otherwise wild-type animals, using the osm-10 promoter , resulted in behavioral hypersensitivity to dilute (1 mM) quinine, similar to rgs-2(lof) and rgs-3(lof) animals, respectively (p>0.05 for both transgenes when compared to the respective rgs loss-of-function animals). The srb-6 promoter was also used for ASH knock-down of rgs-3, and similarly resulted in hypersensitivity to 1 mM quinine (data not shown). (C) Wild-type animals overexpressing ectopic rgs-2 or rgs-3 cDNA displayed diminished response to 10 mM quinine (p<0.0001 when compared to wild-type animals). (D) rgs-2(lof);egl-4(lof) and rgs-3(lof);egl-4(lof) double mutant animals responded to dilute (1 mM) quinine similarly to egl-4(lof) animals (p>0.1 for each). (E) egl-4(gof) animals lacking either RGS-2 or RGS-3 function responded to 10 mM quinine similarly to the rgs-2(lof) and rgs-3(lof) animals, respectively (p>0.5) and (F) were hypersensitive to 1 mM quinine (p<0.001 when compared to wild-type animals). (G) rgs-2(lof) and rgs-3(lof) animals are hypersensitive to dilute (1 mM) quinine. ASH expression of wild-type RGS-2 or RGS-3, using the osm-10 promoter , rescued quinine hypersensitivity in the respective loss-of-function animals. The predicted PKG phosphorylation target site in each was mutated (ΔP), making RGS-2(S126A) and RGS-3(S154A). rgs-2(lof) animals expressing RGS-2(ΔP) and rgs-3(lof) animals expressing RGS-3(ΔP) remained hypersensitive to dilute quinine (p>0.05 for each). The percentage of animals responding is shown. The combined data of ≥3 independent lines, n≥120 transgenic animals, is shown. Error bars represent the standard error of the mean (SEM). Alleles used: egl-4(n479), rgs-1(nr2017), rgs-2(vs17), rgs-3(vs19), rgs-6(vs62), rgs-10(ok1039), rgs-10/11(vs109), egl-10(md176) and eat-16(tm761) loss-of-function and egl-4(ad450) gain-of-function. WT = the N2 wild-type strain. lof = loss-of-function. gof = gain-of-function.

Figure 5. Guanylyl cyclases act upstream of EGL-4.

(A) The osm-10 promoter was used to express wild-type EGL-4 or the cGMP binding mutant EGL-4(T276A) in the ASH sensory neurons of egl-4(lof) animals. While wild-type EGL-4 significantly rescued the egl-4(lof) quinine hypersensitivity (p<0.001), egl-4(lof) animals expressing EGL-4(T276A) remained hypersensitive. (B) Loss-of-function mutations in the guanylyl cyclase genes odr-1, gcy-27, gcy-33 and gcy-34 resulted in behavioral hypersensitivity to dilute (1 mM) quinine (p<0.01 for each). egl-4(gof) animals lacking ODR-1, GCY-27, GCY-33 or GCY-34 function showed diminished sensitivity to both (C) 10 mM quinine and (D) 1 mM quinine, similar to egl-4(gof) single mutant animals (p>0.1 for each double mutant when compared to egl-4(gof) animals). The percentage of animals responding is shown. (E) The osm-10 promoter (ASH, ASI, PHA and PHB), srb-6 promoter (ASH, ADL, ADF, PHA and PHB), and srbc-66 (ASK) promoters were used in cell-selective rescue experiments. The quinine hypersensitivity of odr-1(lof) animals was rescued by srb-6p::odr-1 expression (p<0.001), but not osm-10p::odr-1 expression (p>0.5). gcy-27(lof) hypersensitivity was rescued by all three promoters (p<0.001 for each). Neither gcy-33(lof) nor gcy-34(lof) hypersensitivity was rescued using the osm-10 or srb-6 promoters (p>0.05 for each). The combined data of ≥3 independent lines, n≥120 transgenic animals, is shown. Error bars represent the standard error of the mean (SEM). Alleles used: egl-4(n479), odr-1(n1936), gcy-27(ok3653), gcy-33(ok232) and gcy-34(ok2953) loss-of-function and egl-4(ad450) gain-of-function. WT = the N2 wild-type strain. lof = loss-of-function. gof = gain-of-function.

Figure 6. Model for EGL-4 regulation of nociceptive signaling.

The cGMP-dependent protein kinase EGL-4 regulates C. elegans behavioral sensitivity to the bitter tastants quinine and amodiaquine and the volatile odorant octanol. Wild-type chemosensory signaling is initiated in the ASH sensory neurons when a ligand (such as quinine) binds to a GPCR to activate the associated heterotrimeric G proteins. The activated G proteins (Gα-GTP and Gβγ) interact with downstream effectors to generate second messengers that can activate channels in the plasma membrane, allowing Ca²⁺ influx. Through connections with downstream interneurons and motor neurons, ASH activation is ultimately translated into behavioral avoidance (backward locomotion). Signaling is terminated in part by regulator of G protein signaling (RGS) proteins, which promote the hydrolysis of GTP to GDP by the Gα subunit. EGL-4 phosphorylation of RGS-2 and RGS-3 stimulates their activity. The guanylyl cyclases ODR-1, GCY-27, GCY-33 and GCY-34 may function in alternate neurons to provide the cGMP that is required for EGL-4 function in ASH. In the absence of EGL-4 function, RGS-2 and RGS-3 do not efficiently downregulate Gα signaling, leading to increased Ca²⁺ levels in response to receptor activation. This increased signaling in the ASH sensory neurons leads behavioral hypersensitivity to weak stimuli. The molecular events believed to be happening within the ASHs themselves are included within the grayed area.