Characterization of a novel tyramine-gated chloride channel from Haemonchus contortus

Abstract

Tyramine (TA), a trace amine, is becoming accepted as a main stream neurotransmitter in invertebrates. Recent evidence indicates that part of the function of TA in nematodes involves a novel receptor (Cel-LGC-55) from the ligand-gated chloride channel class of ionotropic receptors. However, the role of TA or its receptors in the biology of nematode parasites is limited. Haemonchus contortus is a deadly parasitic worm which causes significant economic burden in the production of small ruminants in many parts of the world. In this study, we have cloned and characterized a novel LGCC from H. contortus which we have named Hco-LGC-55. This receptor subunit is a clear orthologue of Cel-LGC-55 and is able to form a homomeric chloride channel that is gated by tyramine, dopamine and octopamine. Semi-quantitative reverse transcription-polymerase chain reaction (sqRT-PCR) shows that this subunit is expressed in all life-cycle stages of the worm, but appears to have reduced mRNA expression in the adult male.

Introduction

Infection with the parasitic nematode Haemonchus contortus is one of the biggest challenges in the production of small ruminants causing high economic losses worldwide [1]. H. contortus and the model free-living nematode Caenorhabditis elegans are both members of clade V [2] and as such, many of the proteins that are found in C. elegans appear to have counterparts with similar sequence homology in H. contortus. This is especially true for a very large family of proteins called ligand-gated chloride channels (LGCCs). LGCCs are pentameric membrane proteins that play an important role in fast-synaptic inhibitory neurotransmission [3]. There appears to be several unique LGCC families found in both C. elegans and H. contortus that are activated by a wide range of neurotransmitters such as glutamate [4], [5], GABA [6], and acetylcholine [7]. In addition, recent finding have revealed that both nematodes utilize a novel family of LGCCs that are receptors for biogenic amines, such as serotonin [8], dopamine [9], [10] and tyramine [10], [11]. The fact that these novel amine receptors have only been found in nematodes and not mammals, may position them as novel targets for future anthelmintic development. However, the first endeavor is to clearly define their importance in the biology of parasitic nematodes such as H. contortus. Furthermore, since many of these receptors appear to be common in C. elegans and H. contortus, this will facilitate future investigations that compare the function of these proteins between two nematodes with completely different lifestyles.

TA is a biogenic amine that is more abundant in invertebrate organisms compared with vertebrates [12], and has long been considered as a biosynthetic intermediate of octopamine and not as a neurotransmitter per se. However, recent research on C. elegans indicates that TA is, in fact, a true neurotransmitter. For instance, it has been demonstrated that in C. elegans tyraminergic cells are distinct from octopaminergic cells [13] while TA itself plays a crucial role in the suppression of head oscillations during the worm's response to anterior touch. This action is the direct result of the activation of a novel type of TA-gated chloride channel called Cel-LGC-55 [11]. Furthermore, there are other C. elegans tyramine receptors from the G-protein-coupled receptor class (SER-2, TYRA-2 and TYRA-3) that have also been identified [14], [15], [16], [17]. While these findings have contributed to mounting evidence of the importance of this biogenic amine in C. elegans, there is not much known about the significance of TA as a neurotransmitter in parasitic nematodes. Previous research has demonstrated the presence of TA in the parasite Nippostrongylus brasiliensis [18] and Trichostrongylus colubriformis [19], while TA-receptors from the GPCR class have been identified in Brugia malayi [20]. These findings indicate that, as in C. elegans, TA and its receptors may play important roles in parasitic nematodes. In our study, we have cloned and characterized a novel TA-gated chloride channel (Hco-LGC-55) subunit from H. contortus. This channel responds strongly to TA and dopamine (DA) and to a lesser extent octopamine (OA).