Binding nAChR

Agonists activate nAChR by binding to the agonistbinding site (Fig. 1). They can remain bound (often with higher affinity) when the nAChR enters the desensitised state. 

Non-selective Cation Channels. Figure 1 The nicotinic acetylcholine receptor (nAChR) is localized within the cell membrane above the cell membrane is the synaptic cleft, below the cytoplasm. Drawing of the closed (left) and open (right) nAChR showing acetylcholine (ACh) binding and cation movement. Dimensions of the receptor were taken from references [2, 3]. 

In their search for new hgands with a very high binding affinity for the nicotinic acetylchohne receptor (nAChR), potentially useful in positron emission tomography (PET) when radiolabeled with [ F], Horti et al. described the synthesis of BOC-protected 5-(azetidin-2-ylmethoxy)-2-chloro-6 -fluoro-3,3 -bipyridine via a sequential classical heating and microwave irradiation of (2-fluoro-5-pyridinyl)(trimethyl)stannane with f-butyl 2- [(6-chloro-5-... 

The nAChR subtypes vary in response to pharmacological manipulation. The a7 receptors have a low affinity for nicotine and are sensitive to a-bungarotoxin (a-BTX) antagonism, whereas the heteromeric nAChRs are not.14 The p2 containing (p2 asterisk denotes the presence of additional subunits) nAChRs have the highest affinity for nicotine binding and some selectivity for antagonism... 

Figure 2.1 Diagram of nicotinic acetylcholine receptor (nAChR) structure. A top view of (A) an a7 nAChR and (B) a p2 nAChR shows that homomeric and heteromeric classes of nAChRs are both pentameric in structure. Each subunit is made up of four transmembrane domains with the M2 domain making up the ion pore. (C) A side view of the four transmembrane regions shows the N terminus, C terminus, and large M3-M4 intracellular loop that make up each nAChR subunit. The extracellular loops are available for binding to ligands and the intracellular loop is available for regulation of the nAChR by intracellular signaling proteins.

Figure 3.1 Transaxial parametric images in units of V/ show regional [123I]5-IA binding to p2-nAChR prior to and 2 to 3.5 h and 4 to 5.5 h after smoking a single cigarette. The bar at the left illustrates shades of gray corresponding to V/ values.

The interaction of ACh with the Torpedo nAChR. The data shown compare the equilibrium binding parameters obtained from fluorescence studies using covalently attached fluorescent probes and those obtained from radiolabelled [ H]ACh binding studies or functional measurements of cation flux. These data support a model in which the Torpedo nAChR carries sites of different affinities for ACh. We have previously suggested that occupancy of the lower affinity sites leads to channel activation whereas the higher affinity sites may play a role in desensitization processes... 

To illustrate the principles of an equilibrium dialysis experiment, we will describe the binding of [ H] acetylcholine to the nicotinic acetylcholine receptor (nAChR) in native membranes from Torpedo electro-plax. The data obtained from such an experiment are shown in Figure 10-7 where they are compared with data obtained from a centrifugation assay described below (Protocol 4.2). 

Representative data for [ H]acetylcholine binding to the membrane-bound Torpedo nAChR. Bindng was measured either by equilibrium dialysis (closed circles) as described in Protocol 4.1 or by centrifugation (open squares, see Protocol 4.2). Estimated Kd values from nonlinear regression curve fitting were 12 nM and 10 nM, respectively with corresponding Rq values of 0.14 ulM and 0.135 ulM... 

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