Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle

Abstract

Lymphatic filariasis is a debilitating disease caused by clade III parasites like Brugia malayi and Wuchereria bancrofti. Current recommended treatment regimen for this disease relies on albendazole, ivermectin and diethylcarbamazine, none of which targets the nicotinic acetylcholine receptors in these parasitic nematodes. Our aim therefore has been to develop adult B. malayi for electrophysiological recordings to aid in characterizing the ion channels in this parasite as anthelmintic target sites. In that regard, we recently demonstrated the amenability of adult B. malayi to patch-clamp recordings and presented results on the single-channel properties of nAChR in this nematode. We have built on this by recording whole-cell nAChR currents from adult B. malayi muscle. Acetylcholine, levamisole, pyrantel, bephenium and tribendimidine activated the receptors on B. malayi muscle, producing robust currents ranging from >200 pA to ~1.5 nA. Levamisole completely inhibited motility of the adult B. malayi within 10 min and after 60 min, motility had recovered back to control values.

Keywords: Acetylcholine receptor; Anthelmintic; Brugia malayi.

Figure 1

Whole-cell patch-clamp of B. malayi muscle cells. Top: Micrograph of dissected adult female showing the muscle cells (m) and arms (a) projecting to the nerve cord. Below: Diagramatic representation of the whole-cell patch-clamp.

Figure 2

(A) Bar chart (mean ± s.e) of normalized currents produced by the nAChR agonists/anthelmintics (30 µM) on B. malayi muscles in whole-cell patch-clamp. All current response was normalized to ACh currents (*p < 0.05, paired t-test). Displayed above the bars are sample whole-cell current traces. Scale bar, horizontal 30 secs, vertical 700 pA (B) Plot of motility vs time (min) of adult female B. malayi in the absence and presence of 30 µM levamisole. 4 worms/treatment were used for the assay. Comparisons of motility were made between control and treated worms at each time point, **p<0.01, ***p<0.001.