Comparative pharmacology of rat and human α7 nAChR conducted with net charge analysis

Abstract

1 Pharmacological studies of a7 nicotinic acetylcholine receptors are confounded by the fact that rapid desensitization to high agonist concentration causes a7 peak responses to occur well in advance of complete solution exchange. For this reason, peak currents are an invalid measure of response to applied agonist concentrations. We show that results comparable to those that have been corrected for instantaneous concentration are obtained if net charge is used as the measure of receptor response. 2 Dose response curves obtained with these methods indicate that a7 receptors are approximately 10 fold more sensitive to agonist than previously reported. The agonists, ACh, choline, cytisine, GTS-21, 4OH-GTS-21 and 4-MeO-CA have the same rank order potency for both human and rat receptors: 4-MeO-CA 4 4OH-GTS-21 4 GTS-21 4 cytisine 4 ACh 4 choline. However, di erences in e cacy exist between rat and human receptors. GTS-21 is more e cacious for rat than human a7 receptors and cytisine more e cacious for human than rat a7 receptors. 3 Choline is the least potent agonist for both human and rat a7, with a potency approximately 10 fold lower than that of ACh. While the EC50 for the activation of a7 receptors by choline (400 ± 500 mM) is outside the normal physiological range (10 ± 100 mM), choline can nonetheless produce detectable levels of channel activation in the physiological concentration range. Since these concentrations are relatively non-desensitizing, the contribution of choline-activated a7 receptor current may play a signi®cant role in the regulation of calcium homeostasis in a7-expressing neurons.

Creating an α7 Nicotinic Acetylcholine Recognition Domain from the Acetylcholine-binding Protein

“…5-10-fold differences for the same ligand between animal species are also found. The Ac-AChBP ␤8 -9 linker more closely resembles the rat nAChR than human for homologous residues within binding proximity (27). Thus one can expect similar differences in a comparison of h␣7-nAChR values with those of a soluble receptor surrogate.…”

“…Mean peak FRET ratios calculated from replicates, using SoftMax Pro 5.4 (Molecular Devices) were exported and plotted against agonist concentrations using GraphPad Prism 4. Net charge analysis (27) was not used because the positive allosteric modulator minimizes desensitization, and responses do not necessarily return to base line within the time frame of our experiments. A sigmoidal concentration-response (variable slope) regression of the mean peak FRET ratios was fit to generate a concentration-response curve and obtain an EC 50 value.…”

The α7 nicotinic acetylcholine receptor on fibroblast‐like synoviocytes and in synovial tissue from rheumatoid arthritis patients: A possible role for a key neurotransmitter in synovial inflammation

“…Cycling conditions were 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. As a positive control, cDNA of the human neuroblastoma cell line SKNSH was used (HTB-11; American Type Culture Collection, Teddington, UK) (35). Duplicates were performed for each sample.…”

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD)

“…These ACh control values were chosen because they evoke robust and reproducible responses for each of the respective subtypes, minimizing the chance that rundown or desensitization would be misinterpreted as inhibition. Both the peak amplitude and net charge [69] of the responses were measured for each drug application and calculated relative to the preceding ACh control responses to normalize the data, compensating for the varying levels of channel expression among the oocytes. Net charge values were used to report inhibitory effects.…”