A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

Abstract

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of neurotransmitter-gated ion channels, a family that also includes receptors for γ-aminobutyric acid, glycine and 5-hydroxytryptamine. In humans, nAChRs have been implicated in several neurological and psychiatric disorders and are major targets for pharmaceutical drug discovery. In addition, nAChRs are important targets for neuroactive pesticides in insects and in other invertebrates. Historically, nAChRs have been one of the most intensively studied families of neurotransmitter receptors. They were the first neurotransmitter receptors to be biochemically purified and the first to be characterized by molecular cloning and heterologous expression. Although much has been learnt from studies of native nAChRs, the expression of recombinant nAChRs has provided dramatic advances in the characterization of these important receptors. This review will provide a brief history of the characterization of nAChRs by heterologous expression. It will focus, in particular, upon studies of recombinant nAChRs, work that has been conducted by many hundreds of scientists during a period of almost 30 years since the molecular cloning of nAChR subunits in the early 1980's.

Differential sensitivity of Ctenocephalides felis and Drosophila melanogaster nicotinic acetylcholine receptor α1 and α2 subunits in recombinant hybrid receptors to nicotinoids and neonicotinoid insecticides

“…Taken together, the combined data of our current and earlier studies suggest that insect a1 may play a central role in neonicotinoid insecticide action. However, there should be awareness that the majority of studies were and still are performed with transspecies hybrid receptors, as 'insect-only' nAChRs have not yet been reported in heterologous systems (Millar, 1999(Millar, , 2009, with the exception of a homomeric locust receptor (Marshall et al, 1990;Amar et al, 1995). So far, it is unknown whether expression system factors, such as growth temperature (Lansdell et al, 1997), as yet unrecognized missing subunits, or the need for specific folding factors, such as RIC-3 (Millar, 2008) or Lynx (Liu et al, 2009b), perhaps not present or insufficiently present in the Table 2 Xenopus oocyte coexpression and electrophysiology of D. melanogaster a1 and a2 with chicken b2: EC 50 , Hill coefficient n H and I max .…”

“…Successful examples are largely limited to the formation of trans-species hybrid heteropentamers of insect a subunits with either chicken or rat b2 subunits (Millar, 1999(Millar, , 2009. Xenopus oocyte expression and electrophysiology experiments with hybrid nAChR consisting of D. melanogaster Dma2 and chicken b2 (Ggb2) subunits suggested partial agonist activity of imidacloprid and super-agonist activity of clothianidin (Matsuda et al, 1998;Ihara et al, 2004).…”

“…1) act as partial or full agonists in an in vivo setting. The low toxicity of neonicotinoids to mammals has been explained by their rather poor agonist action on most mammalian nAChRs, as opposed to their strong agonist potency on insect nAChRs (Tomizawa and Casida, 2003, 2005, 2009Matsuda et al, 2001Matsuda et al, , 2005Matsuda et al, , 2009.…”

Allosteric modulation of nicotinic acetylcholine receptors

“…Nevertheless, the combined use of a variety of approaches, including electrophysiological, pharmacological, biochemical and computational techniques can provide strong evidence for the location of ligand binding sites [138].…”

Five nicotinic acetylcholine receptor subunits from the Morotoge shrimp,Pandalopsis japonica: cloning, tissue distribution, and functional expression inXenopusoocytes

“…Functional expression of Pajβ1 subunit with rat α4 subunit on the surface of Xenopus oocytes To determine the subunit composition of the functional P. japonica nAChR channel, all possible combinations of the five nAChR subunit cRNAs were injected into Xenopus oocytes, which is one of the most commonly used systems for functional expression of nAChRs (Millar 2009). Unfortunately, no response was detected to the ACh pulses from any combination of the five isolated nAChR subunits, indicating that no functional channels formed in the Xenopus oocyte system.…”