Rat neuronal nicotinic acetylcholine receptors containing α7 subunit: pharmacological properties of ligand binding and function

Abstract

Aim:To compare pharmacological properties of heterologously expressed homomeric α7 nicotinic acetylcholine receptors (α7 nAChRs) with those of native nAChRs containing α7 subunit (α7* nAChRs) in rat hippocampus and cerebral cortex. Methods: We established a stably transfected HEK-293 cell line that expresses homomeric rat α7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat α7* nAChRs in rat hippocampus and cerebral cortex. We used [ 125 I]-α-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the α7 nAChRs expressed in this cell line were studied using whole-cell current recording. The acetylcholine activated currents were potently blocked by two selective antagonists of α7 nAChRs, α-bungarotoxin (IC 50 =19±2 nmol/L) and methyllycaconitine (IC 50 =100±10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric α7 nAChRs and native α7* receptors in rat brain, but it also revealed several notable differences. Conclusion: This newly established stable cell line should be very useful for studying the properties of homomeric α7 nAChRs and comparing these properties to native α7* nAChRs.

Regulation of Glutamate Release by α7 Nicotinic Receptors: Differential Role in Methamphetamine-Induced Damage to Dopaminergic and Serotonergic Terminals

“…In fact, while acute nicotine pretreatment protected against Meth-induced decreases in [ 3 H]mazindol binding sites in wild-type mice, nicotine had no effect in ␣4 nACh receptor subunit knockout mice (Ryan et al, 2001). In addition, there is evidence that nicotine has a greater affinity for nACh receptors containing the ␣4 subunits (Xiao et al, 2009), suggesting that nicotine affords protection through the activation of nACh receptors containing ␣4 subunits, whereas ACh affects glutamate release and excitotoxicity through the ␣7 receptor. Escubedo et al (2005) suggested that MLA protects against Meth-induced striatal dopaminergic damage in mice through the attenuation of Meth-induced oxidative stress; however the initiation of a pro-oxidant process was not identified.…”

Endogenously Expressed Muscarinic Receptors in HEK293 Cells Augment Up-regulation of Stably Expressed α4β2 Nicotinic Receptors

“…Nicotine-, Carbachol-, and Choline-induced Up-regulation of Other nAChR Subtypes-We also tested nicotine-, carbachol-, and choline-induced up-regulation of several other nAChR subtypes, ␣3␤4, ␣7, ␣4␤4, ␣6␤2, and ␣3␤2 nAChRs stably expressed in HEK293 cells (39,43). Concentrations of nicotine, carbachol, and choline that resulted in nearly maximal or maximal up-regulation of ␣4␤2 receptors after 24 h ( Fig.…”

“…Cell Culture and Treatment-HEK293 cells were maintained and grown as described previously (39,42,43). Cell culturing medium contained Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, and 0.7 mg/ml Geneticin (G418).…”

MD-354 selectively antagonizes the antinociceptive effects of (−)nicotine in the mouse tail-flick assay

“…Stably transfected HEK 293 cells expressing rat α7 neuronal nAChRs were prepared as described previously (Moaddel et al 2008;Xiao et al 2009). The cell line was maintained at 37°C with 5% CO 2 in the incubator.…”

“…The patch electrodes, pulled from borosilicate glass capillaries (Sutter Instrument Company, Novato, CA, USA), had a resistance of 2.5-3.5 MΩ when filled with an internal solution containing 110 mM Tris phosphate dibasic buffer, 28 mM Tris base, 11 mM EGTA, 2 mM MgCl 2 , 0.1 mM CaCl 2 , and 4 mM Mg-ATP (pH adjusted to 7.3 with Tris base; Abdrakhmanova et al 2008;Xiao et al 2009). In some cells, ∼85% of electrode resistance was compensated electronically, so that the effective series resistance in the whole-cell configuration was accepted when less than 20 MΩ.…”