Kinase experiment:

Competition binding reactions used 25 μg human M1 CHO membrane protein, BQCA or vehicle, and 0.15 nM [3H]NMS in 96-well deep-well plates. Binding reactions (30 ℃ for 2-3 h) are terminated by rapid filtration. Nonspecific binding is determined by adding 10 μM atropine. Filter plates are ished 4×with ice-cold 20 mM HEPES, 100 mM NaCl, and 5 mM MgCl2, pH 7.4 using a 96-well harvester. Plates are dried and radioactivity counted with a microplate scintillation counter[1].

Animal experiment:

Rats: Male Sprague-Dawley rats weighing 225-250 g, are injected i.p. with the micro-suspension (containing 10% tween 80) of BQCA at the dose of 10 mg/kg. The blood and whole brain tissue samples are collected at 0.5, 1, 2, 4 and 8 h. Blood samples are collected through cardiac puncture in EDTA vacutainer tubes. The plasma is separated by centrifugation and stored at –80℃ until analysis. The animals are decapitated and the whole brain tissue are removed and immediately frozen on dry ice[2]. Mice: Mice are dosed I.P. with BQCA in 5% beta-cyclodextrin and/or 0.3 mg/kg scopolamine in 0.9% saline 30 min before placement into a chamber for 2 min before 2 tone-footshock pairings (3 kHz, 85 dB tone for 30 s co-terminated with a 0.5 mA, 1 s shock) 2 min apart. Mice are removed to their home cage 30 s after the last pairing. Twenty-four hours later mice are placed into the same chamber and freezing is measured by Video Freeze[1].

Benzyl Quinolone Carboxylic Acid (BQCA) is a highly selective allosteric potentiator of the M1 muscarinic acetylcholine receptor (mAChR) [1].

M1 is most abundantly mAChR expressed in the hippocampus, cortex, and striatum, and localizes to postsynaptic membranes. M1 regulates several ion channels such as KCNQ inwardly rectifying K+currents, voltage-gated calcium channels, and NMDA receptors. M1 mediates the cognitive effects of ACh. M1 activation could slow AD progression by reducing Aβ42 peptides [1].

In vitro: BQCA alone showed no effect on calcium mobilization up to 10 μM but increased ACh potency 128.8 ± 20.1-fold at 100 μM. In CHO cells stably expressing human M1, BQCA (100 μM) activated M1 in the absence of ACh to an approximate 50% maximal response. BQCA had no effect on M2–M5, indicating 100-fold selectivity [1]. BQCA dose-dependently reduced the concentration of acetylcholine required to activate the M1 receptor [1]. The effective range for potentiation of M1 in cells by BQCA was 0.1 to 100 μM, with an inflection point value of 845 nM when 3 nM acetylcholine was used [1].

In vivo: In wild-type mice, BQCA (15 mg/kg) induced c-fos and arc RNA in the cortex, hippocampus, and cerebellum and the arc was also elevated in the striatum. BQCA had no effect in M1-/- mice. In wild-type mice, oral administration of 15 mg/kg BQCA increased the ratio of phosphoERK (pERK) to total ERK by 28%. BQCA showed excellent brain penetration and increased the firing rate of medial prefrontal cortex neurons in vivo in rats [2].

References:
[1] Ma L, Seager M A, Wittmann M, et al.  Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation[J]. Proceedings of the National Academy of Sciences, 2009, 106(37): 15950-15955.
[2] Shirey J K, Brady A E, Jones P J, et al.  A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning[J]. Journal of Neuroscience, 2009, 29(45): 14271-14286.