Pharmacological similarities between native brain and heterologously expressed α4β2 nicotinic receptors

1. We studied the pharmacological properties of native rat brain and heterologously expressed rat α4β2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-α4 antibody mAb 299. 2. Immunodepletion with the anti-β2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained β2. 3. The association and dissociation rate constants for 30 pM ±[(3)H]-epibatidine binding to α4β2 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°C. 4. The Hill coefficients for ±[(3)H]epibatidine binding to the native brain, α4β2 receptors expressed in oocytes, and α4β2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69–0.70 suggesting a heterogeneous receptor population. Fits of the ±[(3)H]-epibatidine concentration-binding data to a two-site model gave K(D) s of 8–30 and 560–1,200 pM. The high-affinity sites comprised 73–74% of the native brain and oocyte α4β2 receptor population, 85% of the CV-1 α4β2 receptor population. 5. The expression of rat α4β2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1.0±0.2). Fits of the ±[(3)H]-epibatidine binding data to a single-site model gave a K(D) of 40±3 pM. 6. DHβE (IC(50)=260–470 nM) and the novel nicotine analogue NDNI (IC(50)=7–10 μM) inhibited 30 pM±[(3)H]-epibatidine binding to the native brain and heterologously expressed α4β2 receptors equally well. 7. The results show that α4β2-containing nicotinic receptors in the rat brain and heterologously expressed rat α4β2 receptors have similar affinities for ±[(3)H]-epibatidine, DHβE, and NDNI