Pharmacological similarities between native brain and heterologously expressed alpha 4 beta 2 nicotinic receptors

1 We studied the pharmacological properties of native rat brain and heterol ogously expressed rat alpha 4 beta 2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha 4 antibody mAb 299. 2 Immunodepletion with the anti-beta 2 antibody mAb 270 showed that 89% of the mAb-299 precipitated rat brain receptors contained beta 2. 3 The association and dissociation rate constants for 30 pM +/-[H-3]-epibat idine binding to alpha 4 beta 2 receptors expressed in oocytes were 0.02 +/ - 0.01 and 0.03 +/- 0.01 min(-1) (+/-standard error, degrees of freedom = 7 - 8) at 20 - 23 degrees C. 4 The Hill coefficients for +/-[H-3]-epibatidine binding to the native brai n, alpha 4 beta 2 receptors expressed in oocytes, and alpha 4 beta 2 recept ors expressed in CV-I cells (using recombinant adenovirus) were 0.69-0.70 s uggesting a heterogeneous receptor population. Fits of the +/-[H-3]-epibati dine concentration-binding data to a two-site model gave K-D s of 8-30 and 550-1,200 pM. The high-affinity sites comprised 73 - 74% of the native brai n and oocyte alpha 4 beta 2 receptor population, 85% of the CV-I alpha 4 be ta 2 receptor population. 5 The expression of rat alpha 4 beta 2 receptors in CV-I cells using vaccin ia viral infection-transfection resulted in a more homogeneous receptor pop ulation (Hill coefficient of 1.0 +/- 0.2). Fits of the +/- [H-3]- epibatidi ne binding data to a single-site model gave a K-D of 40 +/- 3 pM. 6 DH beta E (IC50 = 260 - 470 nM) and the novel nicotine analogue NDNI (IC5 0 = 7 - 10 mu M) inhibited 30 pM+/-[H-3]-epibatidine binding to the native brain and heterologously expressed alpha 4 beta 2 receptors equally well. 7 The results show that alpha 4 beta 2-containing nicotinic receptors in th e rat brain and heterologously expressed rat alpha 4 beta 2 receptors have similar affinities for +/-[H-3]-epibatidine, DH beta E, and NDNI.