Pharmacological characterization of the excitatory ‘Cys‐loop’ GABA receptor family in Caenorhabditis elegans

Abstract

BACKGROUND AND PURPOSEIonotropic GABA receptors are evolutionarily conserved proteins that mediate cellular and network inhibition in both vertebrates and invertebrates. A unique class of excitatory GABA receptors has been identified in several nematode species. Despite wellcharacterized functions in Caenorhabditis elegans, little is known about the pharmacology of the excitatory GABA receptors EXP-1 and LGC-35. Using a panel of compounds that differentially activate and modulate ionotropic GABA receptors, we investigated the agonist binding site and allosteric modulation of EXP-1 and LGC-35. EXPERIMENTAL APPROACHWe used two-electrode voltage clamp recordings to characterize the pharmacological profile of EXP-1 and LGC-35 receptors expressed in Xenopus laevis oocytes. KEY RESULTSThe pharmacology of EXP-1 and LGC-35 is different from that of GABA A and GABA A -ρ receptors. Both nematode receptors are resistant to the competitive orthosteric antagonist bicuculline and to classical ionotropic receptor pore blockers. The GABA A -ρ specific antagonist, TPMPA, was the only compound tested that potently inhibited EXP-1 and LGC-35. Neurosteroids have minimal effects on GABA-induced currents, but ethanol selectively potentiates LGC-35. CONCLUSIONS AND IMPLICATIONSThe pharmacological properties of EXP-1 and LGC-35 more closely resemble the ionotropic GABA A -ρ family. However, EXP-1 and LGC-35 exhibit a unique profile that differs from vertebrate GABA A and GABA A -ρ receptors, insect GABA receptors and nematode GABA receptors. As a pair, EXP-1 and LGC-35 may be utilized to further understand the differential molecular mechanisms of agonist, antagonist and allosteric modulation at ionotropic GABA receptors and may aid in the design of new and more specific anthelmintics that target GABA neurotransmission. AbbreviationsEXP-1, expulsion defective; LGC-35, ligand-gated ion channel

Highly Efficient, Rapid and Co-CRISPR-Independent Genome Editing in Caenorhabditis elegans

“…The C. elegans lgc-35 cDNA was isolated and cloned into the Xenopus laevis expression vector pSGEM (a gift from M. Hollman) as previously described ( Jobson et al 2015 ; Nicholl et al 2017 ). All animal care and experimental procedures followed the guidelines from the National Institutes of Health (NIH) and the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan.…”

“…The standard bath solution for dose-response experiments was Frog Ringer’s: 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 10 mM HEPES (pH = 7.4, NaOH). Standard two-electrode voltage clamp recordings were performed as previously described ( Nicholl et al 2017 ).…”

Advances in our understanding of nematode ion channels as potential anthelmintic targets

“…EXP-1 and LGC-35 subunit have all the characteristic domains of a typical ionotropic GABA receptor subunit but with specific amino-acids in the pore-forming domain that confer cation selectivity ( Beg and Jorgensen, 2003 ). EXP-1 and LGC-35 share 53% sequence identity and express as cation-permeable homomeric channels in Xenopus oocytes that were more sensitive to GABA (EXP-1 EC 50 =26.4 ± 1.08 μM; LGC-35 EC 50 =17 ± 0.71 μM) in comparison to muscimol (EXP-1 EC 50 =250.0 ± 22.0 μM; LGC-35 EC 50 =348.0 ± 17.0 μM) ( Beg and Jorgensen, 2003 ; Nicholl et al, 2017 ). The pharmacological profile of EXP-1 and LGC-35 channels were more similar to GABA A -ρ family than GABA A receptors with notable pharmacological differences that distinguishes them from vertebrate and other nematode GABA channels ( Nicholl et al, 2017 ).…”

Multiple combinations of RDL subunits diversify the repertoire of GABA receptors in the honey bee parasite Varroa destructor

“…In mammals, GABA can also play the role of an excitatory neurotransmitter, and this effect results from the inversion of the chloride gradient that causes cellular depolarization (37). Excitatory GABA receptors have been described in Caenorhabditis elegans in which they play important roles in muscular contraction (38). Conversely, GABA-mediated excitatory neurotransmission has never been observed in insects (39,40), although it has been clearly demonstrated that the D. melanogaster GRD and LCCH3 subunits assemble in cationic ligandgated channels in X. laevis oocytes (19).…”