Pharmacological Assessment of M₁Muscarinic Acetylcholine Receptor-G_q/11Protein Coupling in Membranes Prepared from Postmortem Human Brain Tissue

Abstract

ABSTRACT

Cortical M1 receptor concentration increases without a concomitant change in function in Alzheimer's disease

“…Determining the functional changes in brain M 1 receptor activity (Bymaster, et al , 2003; Salah-Uddin, et al , 2008), particularly in people with mild cognitive impairment (MCI), a precursor stage of AD (Petersen, et al , 1999), is of great interest due to its physiologic relevance to cognitive disease processes. Since the M 1 receptor belongs to a G-protein coupled receptor (GPCR) superfamily, functional assays measuring the nucleotide exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) bound to the G-protein α-subunits provide a method to evaluate GPCR functional responses in brain membrane preparations under pseudo-equilibrium conditions (DeLapp, et al , 1999).…”

“…Currently, most muscarinic receptor assays employ antagonist radioligand binding (Ladner, et al , 1995), [ 35 S]GTPγS protein binding to extrapolate total G-protein activation (Gonzalez-Maeso, et al , 2000; Scarr, et al , 2006), or distal signaling in whole cell based assays (Garro, et al , 2001); however, these methods are limited by either their lack of selectivity for the appropriate GPCR-G protein alpha subunit pair or are inappropriate to measure functional responses in postmortem tissue. To overcome these caveats, a more specific and direct measure of GPCR function was developed using a selective G-protein alpha subunit specific antibody capture [ 35 S]GTPγS binding assay (DeLapp, et al , 1999), which uniquely measures muscarinic G q coupled receptor functional responses in human brain (Salah-Uddin, et al , 2008). As designed, the [ 35 S]GTPγS binding assay predominantly measures M 1 receptor functional responses in cortically derived membranes (Bymaster, et al , 2003).…”

The potent M1 receptor allosteric agonist GSK1034702 improves episodic memory in humans in the nicotine abstinence model of cognitive dysfunction

“…Similarly, GSK1034702 was a potent agonist at hM 1 receptors (pEC 50 =8.1) and displayed at least 100-fold selectivity over hM 2-5 receptors. GSK1034702 also demonstrated partial agonist activity (intrinsic activity y0.6) at rat, marmoset and human native tissue M 1 receptors in a guanosine 5k-O-[c-thio]triphosphate ([ 35 S]GTPcS) binding assay as previously described(Salah-Uddin et al 2008). The agonist activity of GSK1034702 was maintained in post-mortem human cortex from controls and AD patients, as measured using [ 35 S]GTPcS binding (pEC 50 values of 7.0 in control tissue and 7.5 and 7.0 in mild and severe disease tissue samples, respectively).…”

Hippocampal Gαq/11 but not Gαo-coupled receptors are altered in aging

“…The reaction buffer contained 100 mM NaCl, 5 mM MgCl 2 in 50 mM HEPES (pH 7.4). To unmask Gα q/11 activity, membranes were pre-treated with 10 mM N -ethylmaleimide for 30 minutes on ice (Salah-Uddin et al, 2008) and guanosine 5′-diphosphate concentration was 0.1 mM (Delapp et al, 1999; Porter et al, 2002). For the Gα o assay, GDP concentration was 50 mM (Delapp et al, 1999).…”