Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function

Mol Pharmacol. 1998 Aug;54(2):322-33. doi: 10.1124/mol.54.2.322.

Abstract

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • Bridged Bicyclo Compounds, Heterocyclic / pharmacology*
  • Calcium / metabolism
  • Cell Line
  • Dose-Response Relationship, Drug
  • Humans
  • Ligands
  • Nicotine / pharmacology
  • Nicotinic Agonists / pharmacology*
  • Pyridines / pharmacology*
  • Rats
  • Receptors, Nicotinic / drug effects*
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / metabolism
  • Rubidium / metabolism
  • Sodium / metabolism
  • Transfection

Substances

  • Bridged Bicyclo Compounds, Heterocyclic
  • Ligands
  • Nicotinic Agonists
  • Pyridines
  • Receptors, Nicotinic
  • nicotinic receptor subunit alpha3
  • Nicotine
  • Sodium
  • epibatidine
  • Rubidium
  • Calcium