Levamisole receptors: a second awakening

Abstract

Levamisole and pyrantel are old (1965) but useful anthelmintics that selectively activate nematode acetylcholine ion channel receptors; they are used to treat roundworm infections in humans and animals. Interest in their actions has surged, giving rise to new knowledge and technical advances, including an ability to reconstitute receptors that reveal more details of modes of action/resistance. We now know that the receptors are plastic and may form diverse species-dependent subtypes of receptor with different sensitivities to individual cholinergic anthelmintics. Understanding the biology of the levamisole receptors is expected to inform other studies on anthelmintics (ivermectin and emodepside) that act on ion channels.

Figure 1

AChR single-channel currents from Oesophagostomum dentatum using the patch-clamp technique [4,43]. (a) Diagram of the production of the membrane vesicles and patch-pipette used to record the levamisole activated AChR channel currents; (inset) adult female O. dentatum. (b) Examples of levamisole activated channel currents recorded at -75mV and +75mV patch potentials and the current voltage plot showing a linear relationship, zero reversal potential, and slope giving a channel conductance of 42 pS. (c) Channel conductance distribution for the anthelmintic sensitive isolate (wild type) showing the four peaks (fitted using the sum of 4 normal distributions). The peaks illustrate the presence of four subtypes present: G25 pS, G35 pS, G40 pS and G45 pS. In the levamisole-resistant (LEVR) isolate there were only three subtypes present because the G35 pS subtype was missing. In the pyrantel resistant isolate, the four G subtypes were present and included the G 35 pS subtype, but there were fewer channels present in the muscle compared with the anthelmintic sensitive (wild) isolate. (d) Diagram of the different channel subtypes detected and presented by size on a basis of the conductances in O. dentatum; G 35 pS is not present in in levamisole resistant isolates, but present in pyrantel isolates.

Figure 2

Assembly and function of levamisole receptors. C. elegans levamisole sensitive AChR channels in the muscle membrane are formed by five subunits (UNC-38, blue; UNC-29, purple; UNC-63, yellow; LEV-1, green; and LEV-8, red) with normal function being supported by OIG-1, NRA-1, NRA-2, NRA-4, SOC-1, TAX-6, TPA-1, LEV-9 and LEV-10 proteins. Once the levamisole AChR channel opens, calcium enters and its signal is amplified by the ryanodine receptor (UNC-68); the increased calcium then initiates contraction, requiring the proteins UNC-22 and LEV-11. The expression of the levamisole receptor subunits requires RNAs encoding the five subunits together with three C. elegans ancillary proteins involved in AChR assembly (RIC-3), folding (UNC-74) and trafficking (UNC-50) [24, 27, 45]. MicroRNA (miR-1) is also known to regulate and modulate the expression of subunit RNA. Abbreviations: ER, endoplasmic reticulum; SR sarcoplasmic reticulum.

Figure 3

Diagram of possible stoichiometric arrangements of some different AChRs from C. elegans, A. suum and H. contortus and the relative potencies of anthelmintic agonists. (a) The C. elegans levamisole AChR. (b) The C. elegans muscle nicotine AChR. (c) The A. suum AChR where levamisole is more potent than nicotine when more cRNA for UNC-29 than UNC-38 is injected. (d) The A. suum AChR where nicotine is more potent than levamisole when more cRNA for UNC-38 than UNC-29 is injected into the Xenopus oocytes for expression. (e) The H. contortus L-AChR 1 receptor, which is most sensitive to levamisole. (f) The H. contortus L- AChR-2, which is most sensitive to pyrantel [31]. (g) Currents recorded from the Xenopus oocyte expressing H. contortus Hco-L-AChR1 receptor when 100 μM acetylcholine (ACh), 100 μM levamisole (Lev), 100 μM pyrantel (Pyr), 100 μM nicotine (Nic) or 100 μM dimethyphenylpiperazinium (DMPP) are applied. Levamisole is the most potent agonist. (h) Currents recorded from the Xenopus oocyte expressing H. contortus Hco-L-AChR2 receptor when 100 μM acetylcholine (ACh), 100 μM levamisole (Lev), 100 μM pyrantel (Pyr), 100 μM nicotine (Nic) or 100 μM dimethyphenylpiperazinium (DMPP) are applied. Pyrantel produces the biggest peak response.