Muscarinic Receptor Binding

Differentiating between muscarinic receptor subtypes using quantitative autoradiography

Contents 1 Background 2 Binding Affinities 3 Fractional Occupancy 4 Determining Receptor Density 5 See Also

Background

ACh binds metabotrophic muscarinic receptors and ionotropic nicotinic receptors. Both types are present in the mammalian central nervous system (CNS); however, while muscarinic receptors are thought to be involved with a number of CNS functions (e.g. motor control, temperature regulation, memory), there is little evidence for a physiological role of nicotinic receptors in the CNS (See Caulfield, 1998; Caulfield, 1993). There are five subtypes of muscarinic acetylcholine receptors (mAChRs M1-M5) which are coupled to G-proteins (Felder, 1995; Fukudome, 2004; Caulfield 1998; Wess, 2004); the M1, M3, and M5 subtypes are positively coupled to phospholipase-C (PLC), whereas M2 and M4 inhibit adenylyl cyclase (Fukudome, 2004; Exton, 1997; Soreq, 2001; Wei, 1994; McKinney, 1991; Heitz, 1999). There is evidence that M1 and M2 mAChRs are expressed in the hippocampus on both pre- and post-synaptic membranes to various degrees (Levey, 1995; Rouse, 1997); however, a lack of molecules that selectively binds to one subtype over the others, makes it unclear to what degree, density, or ratio each subtype is expressed on pre- and postsynaptic membranes (Servent, 2009). Under most physiological conditions, the specific neurological functions mediated by mAChRs are yet to be determined. There are, however, several studies that have examined the contributions of mAChRs when expressed on either the pre- or postsynaptic membrane. Soreq, et al. (2001) suggests that when M1 is expressed on post-synaptic membranes, it acts to propagate the excitatory impulse. Rather convincing evidence shows that when M2 is expressed on the presynaptic membrane in hippocampal and cortical neurons, is acts as an autoreceptor (inhibiting ACh release) (Rouse, 2000; Soreq, 2001; Machova, 2007; Tucek, 2004; Zhang, 2002). Surprisingly, M2 does not display this function when expressed in the striatum (Zhang, 2002). Thus, several pharmacological and physiological studies have provided a general insight of the trafficking and localization of muscarinic receptors, however, a thorough model of cholinergic regulation is yet to be formulated.

Binding Affinities

Information compiled from several studies examining the pharmakinetics of various muscarinic receptor subtypes yielded these mean pKb of the receptor affinity constants (Caulfield, 1998).

pKb Muscarinic Affinity Constants

*	M1	M2
Pirenzepine:	8.15	6.5
AF-DX 384:	7.4	8.6

pKb = -log Kb

Kb = 10^-pKb

Example: Pirenzepine Kb = 10^-8.15 = 7.08 nM

Kb Muscarinic Affinity Constants

*	M1	M2
Pirenzepine:	7.08 nM	316.23 nM
AF-DX 384:	39.80 nM	2.51 nM

Fractional Occupancy

The law of mass action predicts the fractional receptor occupancy at equilibrium as a function of ligand concentration. Fractional occupancy is the fraction of all receptors that are bound to ligand (NB: Kb = Kd).

Concentrations Used in Study

*	Molar Concentration
Pirenzepine:	10 nM
AF-DX 384:	5 nM

Muscarinic Receptor Subtype Binding Affinity as a Percentage

*	M1	M2
Pirenzepine:	58.5%	3.1%
AF-DX 384:	11.2%	66.6%

Determining Receptor Density

Through quantitative autoradiography, initial figures can be determined using relatively specific ligands for each receptor subtype. Mathematical manipulation will allow one to arrive at a more precise figure for determining receptor concentration values.

In this example the concentrations were determined to be-

M1 = 42.8 fmol/mg

M2 = 23.91 fmol/mg

Thus, the equation would be set up as follows

.585x + .031y = 42.8

.112x + .666y = 23.9

solve for x then y

M1 = x = 71.94

M2 = y = 23.78

See Also

Dissociation constant