Pharmacological similarities between native brain and heterologously expressed alpha4beta2 nicotinic receptors.
1 We studied the pharmacological properties of native rat brain and heterologously expressed rat alpha4beta2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha4 antibody mAb 299. 2 Immunodepletion with the anti-beta2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained beta2. 3 The association and dissociation rate constants for 30 pM +/-[3H]-epibatidine binding to alpha4beta2 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 +/-[3H]epibatidine binding to the native brain, alpha4beta2 receptors expressed in oocytes, and alpha4beta2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69 - 0.70 suggesting a heterogeneous receptor population. Fits of the +/-[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8 - 30 and 560 - 1,200 pM. The high-affinity sites comprised 73 - 74% of the native brain and oocyte alpha4beta2 receptor population, 85% of the CV-1 alpha4beta2 receptor population. 5 The expression of rat alpha4beta2 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 +/-[3H]-epibatidine binding data to a single-site model gave a KD of 40+/-3 pM. 6 DHbetaE (IC50=260-470 nM) and the novel nicotine analogue NDNI (IC50=7-10 microM) inhibited 30 pM+/-[3H]-epibatidine binding to the native brain and heterologously expressed alpha4beta2 receptors equally well. 7 The results show that alpha4beta2-containing nicotinic receptors in the rat brain and heterologously expressed rat alpha4beta2 receptors have similar affinities for +/-[3H]-epibatidine, DHbetaE, and NDNI.
Shafaee
N
N
Division of Biomedical Sciences, University of California, Riverside, California, CA 92521-0121, USA.
Houng
M
M
Truong
A
A
Viseshakul
N
N
Figl
A
A
Sandhu
S
S
Forsayeth
J R
JR
Dwoskin
L P
LP
Crooks
P A
PA
Cohen
B N
BN
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D004079:Dihydro-beta-Erythroidine / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D009538:Nicotine / Q000031:analogs & derivatives / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D011233:Precipitin Tests; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014316:Tritium; D014981:Xenopus
D000911:Antibodies, Monoclonal; D019086:Bridged Bicyclo Compounds, Heterocyclic; C403088:N-n-decylnicotinium; D011725:Pyridines; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins; D014316:Tritium; D004079:Dihydro-beta-Erythroidine; D009538:Nicotine; C082748:epibatidine