Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

Pharmacological characterization of SIB-1765F: a novel cholinergic ion channel agonist.

Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (NAChR), nonselectively activates NAChR limiting its use in elucidating the function of NAChR subtypes. SIB-1765F is a subtype selective NAChR agonist that displaces [3H]-nicotine binding with an IC50 of 4.6 nM and [3H]-cytisine binding with an IC50 of 12.2 nM which is 2000- to 6000-fold lower than its displacement of [3H]-QNB or [125I]-alpha-bungarotoxin. SIB-1765F did not inhibit human or rat cholinesterases or the uptake of [3H]-DA in synaptosomal preparations. SIB-1765F mimicked (-)-nicotine in stimulating [3H]-DA release from rat striatal and olfactory tubercle slices, with EC50 values of 99.6 and 39.6 microM, respectively. Such stimulation was sensitive to mecamylamine and DH beta E. SIB-1765F also released endogenous DA in the striatum and the nucleus accumbens as measured by in vivo microdialysis. SIB-1765F was less efficacious than (-)-nicotine at stimulating [3H]-NE release from rat hippocampal slices; in contrast, SIB-1765F increased [3H]-NE release from rat thalamic and cortical slices with efficacies approaching those of (-)-nicotine. Similar to (-)-nicotine and (+/-)-epibatidine, subcutaneous administration of SIB-1765F increased the turnover rate of dopamine ex vivo both in the striatum and olfactory tubercles in a mecamylamine-sensitive manner. Because the release of striatal DA and hippocampal NE appears to be regulated by distinct NAChR, differential effects of SIB-1765F on striatal DA and hippocampal NE release supports the NAChR subtype selectivity of SIB-1765F compared to (-)-nicotine. This is further demonstrated by observations showing that SIB-1765F has a higher affinity for h alpha 4 beta 2 NAChR relative to h alpha 4 beta 4 NAChRs in displacing [3H]-epibatidine binding and increasing cytosolic CA+2 concentration in cell lines stably expressing h alpha 4 beta 2 or h alpha 4 beta 4.