The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction - PubMed (original) (raw)

The composition of the GABA receptor at the Caenorhabditis elegans neuromuscular junction

Bruce A Bamber et al. Br J Pharmacol. 2005 Feb.

Abstract

1. The unc-49 gene of the nematode Caenorhabditis elegans encodes three gamma-aminobutyric acid type A (GABA(A)) receptor subunits. Two of these, UNC-49B and UNC-49C, are expressed at high abundance and co-localize at the neuromuscular junction. 2. The UNC-49B subunit is sufficient to form a GABA(A) receptor in vitro and in vivo. Furthermore, all loss-of-function unc-49 alleles lack functional UNC-49B. No mutations specifically inactivate UNC-49C. Thus, UNC-49C appears to be dispensable for receptor function; however, UNC-49C has been conserved among different nematode species, suggesting it plays a necessary role. 3. To ascertain whether UNC-49C is part of the GABA(A) receptor in vivo, we performed patch-clamp electrophysiology on C. elegans muscle cells. Sensitivity to GABA, and to the antagonists picrotoxin and pregnenolone sulfate, matched the UNC-49B/C heteromer rather than the UNC-49B homomer, for both exogenous and synaptically-released GABA. 4. The synaptic localization of UNC-49C requires the presence of UNC-49B, indicative of a physical association between the two subunits in vivo. Thus, the in vivo receptor is an UNC-49B/C heteromer. 5. UNC-49C plays a negative modulatory role. Using the rapid ligand-exchange technique in vitro, we determined that UNC-49C causes accelerated receptor desensitization. Previously, UNC-49C was shown to reduce single-channel conductance in UNC-49B/C heteromers. Thus, the function of UNC-49B is to provide GABA responsiveness and localization to synapses, while the function of UNC-49C is to negatively modulate receptor function and precisely shape inhibitory postsynaptic currents.

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Figures

Figure 1

Figure 1

Summary of unc-49 gene structure and mRNA splicing. ‘c–c' refers to the conserved cysteine loop. Vertical bars represent transmembrane domains. Arrow indicates location of the deletion and frameshift mutations that eliminate the UNC-49C subunit in the unc-49 rescuing transgene, used to construct the UNC-49B transgenic strain and the UNC-49B-GFPΔC strain. Exons encoding the N-terminal domain are spliced to one of three alternative sets of exons (A, B, and C) encoding carboxy-terminal GABA binding and transmembrane domains, to produce the UNC-49A, UNC-49B, and UNC-49C cDNAs.

Figure 2

Figure 2

Agonist and antagonist sensitivity of C. elegans muscle cells. (a) GABA concentration–response curves for wild-type worms, UNC-49BC transgenic worms, and UNC-49B transgenic worms. Asterisks mark data points that are significantly different (P<0.05) between UNC-49B, and both the wild type and UNC-49BC. (b) Concentration–response curves for PS inhibition of currents evoked by 300 μ

M

GABA. (c) Concentration–response curves for PTX inhibition of currents evoked by 30 μ

M

GABA. Error bars are s.e.m.

Figure 3

Figure 3

Antagonist pharmacology of the synaptic GABAA receptor in C. elegans muscle. Acetylcholine minis were eliminated by bath application of

D

-tubocurare (500 μ

M

). (a) Traces of GABA minis recorded in the absence (left panel) and presence (middle panel) of 300 μ

M

PS. Plot of relative frequencies of GABA minis in the absence and presence of PS (right panel). (b) GABA minis recorded in the absence (left panel) and presence (middle panel) of 1000 μ

M

PTX. Plot of relative frequencies of GABA minis in the absence and presence of PTX (right panel). The genotype of the worm strain is indicated above each trace. PS is pregnenolone sulfate, PTX is picrotoxin. Error bars are s.e.m.

Figure 4

Figure 4

Synaptic localization of UNC-49C-GFP depends on UNC-49B. (a) Punctate nerve cord fluorescence is observed in worms expressing UNC-49B-GFP in the absence of UNC-49C. (b) No nerve cord-associated fluorescence is observed in worms expressing UNC-49C-GFP in the absence of UNC-49B. Fluorescence is observed on muscle arms and muscle cell membranes. The UNC-49C-GFP construct is localized to synapses in the wild type, which expresses UNC-49B (Bamber et al., 1999).

Figure 5

Figure 5

UNC-49C accelerates desensitization. (a) Outside-out patches pulled from HEK 293 cells expressing UNC-49B homomers (top trace) or UNC-49B/C heteromers (middle trace) were exposed to 10 m

M

GABA for 500 ms using the rapid ligand exchange technique. The line above each trace is the corresponding open tip junction potential, indicating when GABA was applied. Accelerated desensitization of UNC-49B/C heteromers is evident when the two traces are normalized and superimposed (lower panel). (b) Desensitization time constants for UNC-49B homomers and UNC-49B/C heteromers _n_=8 and 10, for UNC-49B homomers and UNC-49B/C heteromers, respectively. _τ_w, _τ_s, and _τ_f are the weighted, slow, and fast decay time constants, respectively. Error bars are s.e.m., asterisk indicates P<0.05 (Student's _t_-test).

Figure 6

Figure 6

Model of interactions at the synapse. UNC-49B is linked to the cytoskeleton by a putative receptor tethering protein that binds to the subunit intracellular domain, and UNC-49C becomes localized to the synapse by physically associating with UNC-49B. The functional role of each subunit is indicated.

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