Biochemistry Of Ligand Gated Ion Channels Important To D

尼古丁乙酰胆碱受体（nAChRs）是尼古丁作用的配体门控离子通道，在尼古丁滥用中起重要作用，了解其神经生物学可能是开发更合理的尼古丁滥用治疗方法的核心。受体-配体相互作用的体外表征是理解配体在体内作用的重要步骤（即解释受体功能研究的结果）。为了分析生理条件下配体与受体的相互作用，建立了37℃条件下的竞争受体实验。该实验使用神经影像学研究分支的工作人员开发并表征的[I-125]-5-碘- a -85380 （5IA）作为alpha4 β 2 nAChRs的高亲和力，选择性放射配体，以确定28种尼古丁激动剂和拮抗剂（9种已建立，8种以前开发，11种最近在我们的化学实验室开发）在23℃和37℃下的亲和力。这些配体的Kd值范围从0.01到25,000 nM。将孵育温度从23℃增加到37℃，导致观察到的Kd值的不同增加（从1.0倍到3.3倍），并对受体-配体相互作用的动力学产生不同的影响。例如，5IA的受体-配体复合物在37℃时的缔合和解离比在23℃时快了约10倍，但[H-3]-胞氨酸结合动力学的速率仅提高了3.5倍。温度对受体结合特性影响的这种可变性表明，生理温度下配体-受体相互作用的表征可以更好地预测配体的体内特性，并且可能对解释α 4 β 2 nAChRs功能研究的结果很重要。雅培实验室（Abbott Laboratories）先前的研究表明，Me-p-PVC（5-（2-（4-吡啶基）乙烯基）-6-氯-3-（1-甲基-2-(S)-吡啶基甲氧基）吡啶）对含有alpha3beta4 nachr的烟碱乙酰胆碱受体（nachr）和尼古丁诱导的86Rb从IMR-32细胞外排具有高亲和力。与Me-p-PVC的体外结合研究表明，其对alpha4beta2 nAChRs的亲和力超过了对alpha3beta4受体亚型的亲和力3000倍（Kd分别约为0.025和90 nM）。

Nicotinic acetylcholine receptors (nAChRs), the ligand gated ion channels at which nicotine acts, play an important role in nicotine abuse and understanding its neurobiology may be central to developing more rational approaches for nicotine abuse treatments. The in vitro characterization of the receptor-ligand interaction is an important step in understanding the action of a ligand in vivo (i.e., for interpretation of the results from the functional studies of receptors). To analyze the ligand-receptor interaction at physiological conditions, a competition receptor assay at 37 degrees C was developed. This assay used [I-125]-5-iodo-A-85380 (5IA), which was developed and characterized by staff of the Neuroimaging Research Branch as a high affinity, selective radioligand for alpha4 beta2 nAChRs, to determine the affinities of 28 nicotinic agonists and antagonists (9 established, 8 previously and 11 recently developed in our chemistry lab) at 23 degrees C and 37 degrees C. The Kd values for these ligands ranged from 0.01 to 25,000 nM. Increasing incubation temperature from 23 to 37 degrees C resulted in variable increases in the observed Kd values (from 1.0 to 3.3-fold) and variable effects on the kinetics of receptor-ligand interaction. For example, the association and dissociation of the receptor-ligand complex for 5IA were approximately 10 times faster at 37 degrees C than that at 23 degrees C, but the rate of [H-3]-cytisine binding kinetics increased only 3.5 fold. This variability in effects of temperature on receptor binding properties suggests that characterization of ligand-receptor interaction at physiological temperature may better predict the in vivo properties of the ligands and may be important in the interpretation of results from the functional studies of alpha4 beta2 nAChRs. Previous studies from Abbott Laboratories, where Me-p-PVC (5-(2-(4-pyridinyl)vinyl)-6-chloro-3-(1-methyl-2-(S)-pyrrolidinylmethoxy)pyridine) was developed, demonstrated that this compound had high affinity for alpha4beta2 nicotinic acetylcholine receptors (nAChRs) and attenuated nicotine-induced efflux of 86Rb from IMR-32 cells, contained alpha3beta4 nAChRs. In vitro binding studies with Me-p-PVC showed that its affinity for alpha4beta2 nAChRs exceeded that for the alpha3beta4 receptor subtype by a factor of 3,000 (Kd ca. 0.025 and 90 nM, respectively). Substantial anecdotal evidence suggests that nicotine may function as a gateway drug to illicit drug use. As such we utilized an animal behavorial model of drug rewards conditioned place preference (CPP) to test the hypothesis that nicotine produces behavioral cross-sensitization to different classes of abused drugs, specifically opiates, stimulants and marihuana. These experiments in rats demonstrated that nicotine pretreatment enhances the rewarding effects of amphetamine for at least 3 to 5 days following the cessation of nicotine, with this effect dissipating within 19 days. The underlying mechanism involves alpha4beta2 nAChRs as the competitive alpha4beta2 antagonist dihydro beta erythroidine effectively blocked the development of nicotine-induced cross sensitization. Interestingly, the alpha7 nicotinic antagonist methyllycaconitine also antagonized cross-sensitization at doses that do not block nicotine self-administration in rats. This study and published report have clearly demonstrated that nicotine produces cross-sensitization to the rewarding effects of both opiates and psychostimulants measured with CPP. The development of cross-sensitization to the rewarding effects of these drugs involves the interaction of nicotine with both alpha4beta2 and alpha7 nAChRs, which differs from nicotine self-administration, which is primarily mediated by alpha4beta2 receptors.