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NEUROPHARMACOLOGY

Heterologous Expression of Human 6435 Nicotinic Acetylcholine Receptors: Binding Properties Consistent with Their Natural Expression Require Quaternary Subunit Assembly Including the 5 Subunit

Targacept, Inc., Winston-Salem, North Carolina (V.P.G., S.R.L., K.A.S., M.B.); Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (K.A.L., L.M.B., R.J.L.); and Aventis Pharma SA, Vitry Sur Seine, France (J.M., V.M., G.A.B., L.P., J.B.)

Heterologous expression and lesioning studies were conducted to identify possible subunit assembly partners in nicotinic acetylcholine receptors (nAChR) containing 6 subunits (6^* nAChR). SH-EP1 human epithelial cells were transfected with the requisite subunits to achieve stable expression of human 62, 64, 623, 643, or 6435 nAChR. Cells expressing subunits needed to form 6435 nAChR exhibited saturable [³H]epibatidine binding (K_d = 95.9 ± 8.3 pM and B_max = 84.5 ± 1.6 fmol/mg of protein). The rank order of binding competition potency (K_i) for prototypical nicotinic compounds was -conotoxin MII (6 nM) > nicotine (156 nM) methyllycaconitine (200 nM) > -bungarotoxin (>10 µM), similar to that for nAChR in dopamine neurons displaying a distinctive pharmacology. 6-Hydroxydopamine lesioning studies indicated that 3 and 5 subunits are likely partners of the 6 subunits in nAChR expressed in dopaminergic cell bodies. Similar to findings in rodents, quantitative real-time reverse transcription-polymerase chain reactions of human brain indicated that 6 subunit mRNA expression was 13-fold higher in the substantia nigra than in the cortex or the rest of the brain. Thus, heterologous expression studies suggest that the human 5 subunit makes a critical contribution to 6435 nAChR assembly into a ligand-binding form with native 6^*-nAChR-like pharmacology and of potential physiological and pathophysiological relevance.

Address correspondence to: Merouane Bencherif, Targacept, Inc., 200 East First Street, Suite 300, Winston-Salem, NC 27101. E-mail: merouane.bencherif{at}targacept.com

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