Ligand-gated chloride channels are receptors for biogenic amines in C. elegans

Abstract

Biogenic amines such as serotonin and dopamine are intercellular signaling molecules that function widely as neurotransmitters and neuromodulators. We have identified in the nematode Caenorhabditis elegans three ligand-gated chloride channels that are receptors for biogenic amines: LGC-53 is a high-affinity dopamine receptor, LGC-55 is a high-affinity tyramine receptor, and LGC-40 is a low-affinity serotonin receptor that is also gated by choline and acetylcholine. lgc-55 mutants are defective in a behavior that requires endogenous tyramine, which indicates that this ionotropic tyramine receptor functions in tyramine signaling in vivo. Our studies suggest that direct activation of membrane chloride conductances is a general mechanism of action for biogenic amines in the modulation of C. elegans behavior.

Figure 1. Biogenic amines evoke currents in Xenopus oocytes expressing LGC-53, LGC-55 or LGC-40

A, C, E. Whole-cell currents recorded from Xenopus oocytes expressing LGC-53, LGC-55 or LGC-40 during application of serotonin (5-HT), dopamine (DA), octopamine (OA), tyramine (TA) and histamine (HA). The concentrations of applied neurotransmitters were 10 μM in panels (A) and (B) and 100 μM in (C). B, D. Dose-response curves for dopamine and tyramine-evoked currents in Xenopus oocytes expressing LGC-53 and LGC-55, respectively. Each point (± s.e.m.) represents the average of three to five recordings. Dose-response data were fitted to the Hill equation and normalized to I_max. The half-effector concentration (EC50) of dopamine for LGC-53 was 4.4 μM, and the estimated Hill coefficient was 1.9. The half-effector concentration of tyramine for LGC-55 was 6.0 μM, and the estimated Hill coefficient was 1.8. F. Dose-response curves for serotonin, acetylcholine and choline-evoked currents in Xenopus oocytes expressing LGC-40. Dose-response data were fitted to the Hill equation and normalized to I_max. n ≥ 5. The half-effector concentration of serotonin for LGC-40 was 905 μM, and the estimated Hill coefficient was 2.7. The half-effector concentrations of acetylcholine and choline for LGC-40 were 87 μM and 3.4 μM, respectively, and the estimated Hill coefficients were 1.5 and 1.9, respectively.

Figure 2. Pharmacological characterizations of the ionotropic dopamine receptor LGC-53 and the ionotropic choline receptor LGC-40

A. Inhibition of LGC-53 currents by dopamine-receptor antagonists. The mean ratios (± s.e.m.) of the peak currents evoked in the presence and absence of the indicated dopamine receptor antagonist are shown. n ≥ 5. Drugs were tested at a concentration of 100 μM, and 5 μM dopamine was used to evoke LGC-53 currents. B-D. Dose-response curves for the inhibiton by haloperidol, risperidone, and spiperone of LGC-53 currents. 5 μM dopamine was used to evoke currents in oocytes expressing LGC-53 in the presence of different concentrations of dopamine receptor antagonists. Currents were normalized to the current evoked by 5 μM dopamine in the absence of receptor antagonists. n ≥ 5. E-F. Dose-response curves for the inhibition by atropine, d-tubocurarine, and hemicholinium-3 of LGC-40 currents. Currents were evoked by 2 μM choline in oocytes expressing LGC-40 in the presence of different concentrations of compounds. Currents were normalized the current evoked by 2 μM choline in the absence of any compounds. n ≥ 5.

Figure 3. LGC-40, LGC-53 and LGC-55 are chloride channels

A. Alignment of sequences from the presumptive M2 region of amine-gated ion channels, which determines ion selectivity of Cys-loop family ion channels (9, 12). Cation-selective 5-HT3 receptor subunits are labeled in green, and anion-selective MOD-1, HCLA, and HCLB subunits are labeled in red. B-D. I-V curves of LGC-40, LGC-53 and LGC-55 in ND96 medium (which contains 96 mM sodium and 104 mM chloride), sodium-free medium (0 mM sodium and 104 mM chloride) or low-chloride medium (96 mM sodium and 8 mM chloride). The mean reversal potential ± s.e.m. from four to five experiments under each condition is shown.

Figure 4. LGC-55 is required for the tyraminergic modulation of head movements by C. elegans and is expressed in the GLR glia-like cells and head muscles

A. The fractions of animals that are suppression of head oscillations-defective (Sho) are plotted for the wild type, tdc-1, and lgc-55 mutants and lgc-55 mutants carrying rescuing transgenes that express the lgc-55 cDNA using its own promoter, the pan-neuronal unc-119 promoter, or the pan-muscle myo-3 promoter. In each experiment we tested 20 individuals of each genotype for the Sho phenotype. The mean fraction of animals with the Sho phenotype ± s.e.m. is plotted, n ≥ 3. Three independent lines (labeled #1-3) carrying each transgene were assayed. B. Expression of lgc-55. An lgc-55∷gfp reporter transgene is expressed in the GLR glia-like cells and head muscles (16). Arrowheads indicate some of the unidentified head neurons that express the reporter transgene. The tyraminergic RIM neurons, which provide the tyramine that inhibits head movements during reversals, are labeled with a tdc-1∷dsRed reporter transgene.