Characterization of a macrocyclic lactone receptor subunit from Haemonchus contortus

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics, such as IVM, and the milbemycins, such as MOX.The objective of this thesis was to determine whether Haemonchus contortus GluCls are important targets for these anthelminthics.To begin to address this we cloned a fun length GluCl alpha-subunit cDNA from H. con tortus (HcGluCla).This subunit shares a high homology with GluCl subunits from Caenhorhabditis elegans that have been shown to be important targets for IVM, suggesting that HcGluCla is also an IVM target.However, if HcGluCla is an IVM receptor then it should contain an IVM binding site.To investigate this, the HcGluCla gene was expressed in COS-7 ceUs.The resulting subunit bound CH]IVM and eH]MOX with affinities sufficiently high enough to explain their high in vivo potency.Interestingly, glutamate was an allosteric modulator of eH]MOX and eH]IVM binding where it increased the affinity of these drugs to HcGluCla.To gain further insight into the potentiation of IVM, various glutamatergic and non-glutamatergic ligands were screened for their ability to enhance CH]IVM binding to HcGluCla.Of the ligands tested, only the GluCl agonists glutamate and ibotenate potentiated eH]IVM binding.It is possible therefore that if IVM interacts with GluCls in vivo then IVM efficacy may be enhanced by GluCl agonists.To examine this, we tested wh ether ibotenate could enhance IVM efficacy in gerbils infected with H. contortus.In in vivo efficacy studies, ibotenate (at 1 mg/kg) increased IVM efficacy by 15% (p = 0.048).The enhancement of IVM efficacy in vivo by a GluCl agonist suggests that one of the IVM targets in H. contortus is the GluCl.Finally, to determine the potential physiological response from an interaction between IVM and H. contortus GluCls, we expressed HcGluCla in Xenopus oocytes.HcGluCla expressed in oocytes formed a homomeric channel that responded to glutamate and ibotenate as weIl as the anthelmintics IVM and MOX.The response to glutamate and ibotenate was fast acting and reversible whereas the response to IVM and MOX was a slower activating current that was essentially irreversible.The results from this thesis provide strong evidence that IVM toxicity in H. contortus is the result of its irreversibleactivation of GluCk IV ACKNO'VLEDGEMENTS 1 would like to express my sincere gratitude to my advisors, Drs.Robin Beech and Roger Prichard for giving me the opportunity to study at the Institute of Parasitology. 1 am grateful for their mentorship, financial resources and giving me the freedom to pursue many of the ideas and sometimes the tangents 1 chose to investigate.1 am grateful to many of my lab mates who made the stresses of lab work more bearable, especially Meredith, Cindy and Dr

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