Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor

Abstract

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligand-gated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 alpha- and 2 non-alpha-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.

Fig. 1.

Expression of functional AChRs from C. elegans in X. laevis oocytes. (A) A single oocyte coinjected with the 8 cRNAs unc-29, unc-38, unc-63, lev-1, lev-8, ric-3, unc-50, and unc-74 displays large inward currents elicited by ACh (100 μM) or levamisole (Lev, 100 μM) but not by nicotine (Nic, 100 μM). (B) Coexpression of 5 receptor subunits (black squares) and 3 ancillary factors (gray squares) is mandatory for robust expression of the L-AChR. Currents were measured at the peak. Average peak current for coinjection of all 8 cRNAs was 4.1 ± 3.7 μA (n = 49). (C) A single oocyte coinjected with the acr-16 and ric-3 cRNAs displays large transient inward currents elicited by ACh (500 μM) or nicotine (500 μM), but not by levamisole (500 μM). (D) Functional expression of the homopentameric ACR-16 nicotine-sensitive AChR requires the ancillary factor RIC-3 but not UNC-50 or UNC-74. Currents were measured at the peak. Average peak current for coinjection of acr-16 and ric-3 cRNAs was 6 ± 4.2 μA (n = 33). All recordings were made with 1 mM external CaCl₂. Numbers above bars represent the number of oocytes recorded for each condition.

Fig. 2.

The L-AChR is permeable to calcium and shows no macroscopic desensitization. (A) All traces are from a single oocyte expressing L-AChRs. In 1 mM external CaCl₂, the response to ACh displays a preeminent inward peak current. Chelating intracellular calcium by injection of BAPTA or replacing extracellular calcium by a low amount of barium (0.3 mM) eliminates this peak and results in stable responses upon continuous application of ACh. (Inset) N-AChRs display profound macroscopic desensitization upon continuous application of ACh (500 μM) even after BAPTA injection (n = 6). (B) Comparison of the I/V relationships of L-AChR responses elicited by 100 μM ACh in the presence of 1 mM or 10 mM extracellular CaCl₂. Note that the L-AChR is potentiated by 10 mM extracellular calcium and that this potentiation occurs over the whole voltage range (n = 5, BAPTA-loaded oocytes). (C) Magnified view of the dash-boxed region in B showing the rightward shift of the reversal potential induced by switching from 1 mM to 10 mM external Ca²⁺ (1.6 mV to 3.4 mV for this cell).

Fig. 3.

Agonist pharmacology of the L-AChR. (A) (Left) Acetylcholine, levamisole, and pyrantel, but not nicotine, activate the L-AChR. Concentrations of agonist are indicated above each application. Traces are from a single oocyte. (Right) Current relative to 100 μM ACh (plateau values): 100 μM levamisole 38 ± 6% (n = 6), 100 μM pyrantel 6.1 ± 0.7% (n = 6), 500 μM nicotine 0.55 ± 0.21% (n = 4). (B) Dose–response curves for ACh and levamisole. The value at 500 μM levamisole has been excluded from the fit because of voltage-dependent block at this concentration. (C) High concentrations of levamisole cause voltage-dependent channel block of L-AChR. BAPTA-injected oocytes were subjected to voltage ramps in the presence of 100 μM or 500 μM levamisole (n = 5). (Inset) Representative traces of L-AChR responses evoked by 100 μM and 500 μM levamisole. The rebound of the current after washout of 500 μM levamisole probably occurs because unbinding of levamisole from its pore-blocking site is faster than unbinding of levamisole from its agonist site. (D) Concentration-dependent washout kinetics of levamisole-evoked responses. ACh or levamisole traces obtained from a single oocyte at different agonist concentrations were first normalized to the current level measured just before agonist washout and then superimposed. Note that although ACh-evoked responses display classical concentration-independent washout kinetics, the washout time course of levamisole-evoked responses increases with increasing levamisole concentrations.

Fig. 4.

Antagonist pharmacology of the L-AChR. L-AChRs activated by 100 μM ACh are inhibited by 100 μM dTC (96 ± 1%; n = 7), 10 μM MLA (39 ± 8%; n = 5), and 100 μM Hex (30 ± 2%; n = 5). In contrast, 100 nM α-BgTx and 10 μM DHβE produce only very weak inhibition (7 ± 1%, n = 5; and 6 ± 1%, n = 5; respectively).

Fig. 5.

Nicotine acts mainly as an allosteric inhibitor of the L-AChR. (A) Nicotine fails to activate L-AChR significantly in vivo. In whole-cell recordings from C. elegans body-wall muscles, a strain lacking both acr-16 and unc-13 shows no response to nicotine, unlike unc-13 mutants [mean amplitudes of the response to 500 μM nicotine were 21 ± 11 pA (n = 4) and 420 ± 80 pA (n = 4), respectively]. (B) In contrast, responses to levamisole are indistinguishable between these 2 strains [mean amplitudes of the response to 500 μM levamisole were 155 ± 37 pA (n = 4) and 134 ± 22 pA (n = 4), respectively]. (C) Responses elicited by ACh are inhibited by nicotine in a dose-dependent manner. Percentage inhibition of responses elicited by 100 μM ACh were 26 ± 2% (n = 6) for 100 μM nicotine and 93 ± 3% (n = 6) for 500 μM nicotine. (D) Nicotine inhibition is not voltage-dependent. Voltage ramps (−70 to +50 mV) in 100 μM ACh with or without 500 μM nicotine. Note that nicotine inhibition occurs over the whole voltage range (n = 10, BAPTA-injected oocytes). (E) Nicotine has a modest effect on ACh sensitivity. ACh dose–response curves were performed with or without 300 μM nicotine. In the absence of nicotine, EC₅₀ and n_H for ACh are 26 ± 3 μM and 1.05 ± 0.06 (n = 6–13), respectively. In the presence of nicotine, EC₅₀ and n_H for ACh are 58 ± 3 μM and 1.23 ± 0.06 (n = 5), respectively.