Atomic force microscopy to characterize binding properties of α7-containing nicotinic acetylcholine receptors on neurokinin-1 receptor-expressing medullary respiratory neurons.
Atomic force microscopy to characterize binding properties of α7-containing nicotinic acetylcholine receptors on neurokinin-1 receptor-expressing medullary respiratory neurons.
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DOI：
10.1113/expphysiol.2012.067660
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发表时间：
2013-02
影响因子：
2.7
通讯作者：
Potts JT
中科院分区：
文献类型：
--
作者：
Clark CG;Sun Z;Meininger GA;Potts JT
在本研究中，我们使用原子力显微镜（AFM）检查的配体结合特性的α7-含烟碱乙酰胆碱受体（nAChRs）的腹侧呼吸组的神经元上表达。我们还确定了急性和长期暴露于尼古丁对nAChRs结合概率的影响。培养新生（P5-P10）和幼年（3-4周）大鼠的神经元。Alexa Fluor 488结合的P物质的内化用于鉴定表达神经激肽-1受体（NK 1-R）的呼吸神经元; NK 1-R是腹侧呼吸组神经元的公认标记物。为了评估nAChR的功能变化，使用与抗α7亚基nAChR抗体缀合的AFM探针循环地与NK 1-R阳性神经元的索马表面相互作用。测量了抗体粘附到α7-受体亚基的频率以及膜附着受体与AFM探针尖端之间的分离力。向细胞浴中加入α-银环蛇毒素（含α-7亚基的nAChR的特异性拮抗剂），与α-7亚基的结合减少69%（P<0.05，n=10），支持结合的特异性。急性暴露于尼古丁（1 μM添加到培养基中）使nAChR抗体与α-7亚基的结合减少80%（P<0.05，n=9）。细胞培养物在尼古丁中的长时间孵育（72小时）以浓度依赖性方式显著降低α-7结合。总的来说，这些研究结果表明，AFM是一个敏感的工具，用于评估的nAChRs表达的活性NK 1-R表达髓神经元的表面上的功能变化。此外，这些数据表明尼古丁暴露降低了含nAChR的α-7亚基的结合概率。
In the present study we used atomic force microscopy (AFM) to examine the ligand binding properties of α7-containing nicotinic acetylcholine receptors (nAChRs) expressed on neurons from the ventral respiratory group. We also determined the effect of acute and prolonged exposure to nicotine on the binding probability of nAChRs. Neurons from neonatal (P5–P10) and juvenile (3–4 wk) rats were cultured. Internalization of Alexa Fluor 488–conjugated substance P was used to identify respiratory neurons that expressed neurokinin-1 receptors (NK1-R); a recognized marker of ventral respiratory group neurons. To assess functional changes in nAChRs, AFM probes conjugated with anti-α7 subunit nAChR antibody were used to cyclically interact the soma surface of NK1-R positive neurons. Measurements were made of the frequency of antibody adhesion to the α7-receptor subunit and of the detachment forces between the membrane attached receptor and the AFM probe tip. Addition of α-bungarotoxin (a specific antagonist of α-7 subunit-containing nAChRs) to the cell bath produced a 69% reduction in binding to the α-7 subunit (P<0.05, n=10), supporting specificity of binding. Acute exposure to nicotine (1 μM added to culture media) produced an 80% reduction in nAChR antibody binding to the α-7 subunit (P<0.05, n=9). Prolonged incubation (72 hours) of cell culture in nicotine significantly reduced α-7 binding in a concentration-dependent manner. Collectively, these findings demonstrate that AFM is a sensitive tool for assessment of functional changes in nAChRs expressed on the surface of living NK1-R expressing medullary neurons. Moreover, these data demonstrate that nicotine exposure decreases the binding probability of α-7 subunit containing nAChRs.
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影响因子：
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通讯作者：
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