LGICs

Fig. 1. Model of a ligand gated ion channel (LGIC) where (a) is the structure of a generic LGIC subunit showing the two cysteine (Cys) residues common to all LGIC subunits, and (b) shows the arrangement of five such subunits as a pentamer having psuedo-cyclic symmetry delineating a gated, fluid-filled...

Acetylcholine. Acetylcholiae (ACh) (1) is a crystalliae material that is very soluble ia water and alcohol. ACh, synthesized by the enzyme choline acetyltransferase (3), iateracts with two main classes of receptor ia mammals muscarinic (mAChR), defiaed oa the basis of the agonist activity of the alkaloid muscarine (4), and nicotinic (nAChR), based on the agonist activity of nicotine (5) (Table 1). m AChRs are GPCRs (21) n AChRs are LGICs (22). 

Classification of P2 purinoceptors has been limited by a lack of potent, selective, and bioavailable antagonists. Nonetheless a rational scheme for P2 purinoceptor nomenclature divides P2 receptors into two superfamilies P2Y5 LGIC family having four subclasses and P2Y) a GPCR family having seven subclasses. A third receptor type, designated the P22) is a nonselective ion pore. 

Synaptic Transmission. Figure 1 Synaptic transmission. The presynaptic terminal contains voltage-dependent Na Superscript and Ca2+ channels, vesicles with a vesicular neurotransmitter transporter VNT, a plasmalemmal neurotransmitter transporter PNT, and a presynaptic G protein-coupled receptor GPCR with its G protein and its effector E the inset also shows the vesicular H+ pump. The postsynaptic cell contains two ligand-gated ion channels LGIC, one for Na+ and K+ and one for Cl-, a postsynaptic GPRC, and a PNT. In this synapse, released transmitter is inactivated by uptake into cells. 

Of the several classes of receptors for endogenous chemical signals [3], two are used as postsynaptic receptors in synaptic transmission ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs Fig. 1). Due to the large number of transmitters and the existence of several receptor types for almost all, postsynaptic receptor activation is the most diversified step of synaptic transmission. Table 1 shows selected neurotransmitter receptors. 

All these postsynaptic events last only for a few milliseconds synaptic transmission through LGICs is fast. When the postsynaptic cell membrane is sufficiently depolarized, voltage-dependent Na+ channels open and an action potential is generated. 

Glutamate AMPA (LGIC) t Na+ and K+ conductance a-Am ino-3-hyd roxy-5-methyl-4-isoxazolepropionic acid (AMPA) ... 

GABA GABAa (LGIC) t Cl" conductance Muscimol Bicuculline... 

Glycine glycine receptor (LGIC) f Cl" conductance Strychnine... 

Acetylcholine nicotinic (LGIC) Na+, K+ and Ca2 + conductance Nicotine, suxamethonium Tubocurarine, a-conotoxins, a-bungarotoxin... 

Expression of Functional LGIC Receptors in Xenopus Oocytes. 333... 

High levels of protein expression are generally achieved with cRNA injection. This technique requires the in vitro synthesis of the appropriate cRNA from the template cDNA (see later). Although this approach can be time-consuming and costly, it remains the most common technique used to ensure the robust expression of receptors and ion channels in the oocyte membrane. In our laboratory, we routinely use the cRNA injection technique to promote high expression levels of LGIC receptors (GABAa receptor subtypes, nACh and 5-HT3 receptors). 

With respect to a multi-subunit LGIC receptor, a common approach is to inject oocytes with an equivalent molar ratio of each transcript. However, it should be noted that the ratio of injected transcripts might have an effect on the final stoichiometry of subunits in the expressed receptors (e.g., Boileau et aL, 2002). This may lead to an expression of a heterogeneous population of receptors that can complicate functional analysis. 

Characterization of the properties of LGIC receptors expressed in Xenopus oocytes has heen facihtated hy the variety of studies that can be carried out on recombinant receptors in this system. 

A range of experiments can characterize LGICs that are expressed in Xenopus oocytes. Application of a drug solution into the perfusion system allows for exposure of the oocyte to that concentration of ligand. In these studies, it is usually assumed that the bath volume is relatively small and that there is minimal dead-space in the flow tubing. 

The Xenopus oocyte can reliably express LGIC receptors. In our laboratory, we have seen robust expression of the Torpedo nAChR, 5-HT3 receptors and various GABAa receptor subtypes in oocytes. Injection of cRNA transcripts is a convenient and reproducible way to achieve the expression levels needed for functional analysis of receptor subtypes. We have found that functional characterization with this system complements biochemical experiments conducted on native receptors or those that have been expressed in mammahan cells. A combination of these approaches is essential for furthering our understanding of structure-function relationships in these receptors. 

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