Guangxi Key Laboratory of Special Biomedicine, School of Medicine, Guangxi University, Nanning 530004, China.
Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University, Haikou 570228, China.
Mar Drugs. 2024 Apr 27;22(5):200. doi: 10.3390/md22050200.
nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The nAChR has high Ca permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat nAChRs expressed in oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of nAChRs in the rat brain.
烟碱型乙酰胆碱受体 (nAChRs) 主要分布于中枢神经系统 (CNS)，包括海马体、纹状体和大脑皮层。nAChR 具有较高的钙通透性，能够快速激活和脱敏，与阿尔茨海默病 (AD)、癫痫、精神分裂症、肺癌、帕金森病 (PD)、炎症等疾病密切相关。海洋锥形蜗牛毒液中的α-芋螺毒素通常是针对 nAChR 的短而富含二硫键的神经肽，能够区分各种亚型，为 nAChR 的功能研究提供了重要的药理学工具。[Q1G，ΔR14]LvΙB 是一种大鼠 nAChRs 选择性拮抗剂，由α-芋螺毒素 LvΙB 修饰而来。本研究利用 N-羟基琥珀酰亚胺 (NHS) 激活处理后的三种荧光素：6-TAMRA-SE、Cy3 NHS 和 BODIPY-FL NHS，在弱碱性条件下标记 [Q1G，ΔR14]LvΙB 的 N 端，得到三种荧光类似物：LvIB-R、LvIB-C 和 LvIB-B。利用双电极电压钳（TEVC）技术，评估了[Q1G，ΔR14]LvΙB 荧光类似物在大鼠 nAChRs 表达的卵母细胞中的效力。结果显示，LvIB-R、LvIB-C 和 LvIB-B 的半最大抑制浓度（IC）值分别为 643.3 nM、298.0 nM 和 186.9 nM。脑脊液分析稳定性研究表明，孵育 12 h 后，三种荧光类似物的保留率分别为 52.2%、22.1%和 0%。[Q1G，ΔR14]LvΙB 荧光类似物被应用于探索大鼠脑组织中海马体和纹状体中 nAChRs 的分布情况，结果发现 Cy3 和 BODIPY FL 标记的 [Q1G，ΔR14]LvΙB 比 6-TAMARA- 具有更好的成像特性。还发现 nAChRs 广泛分布于大脑皮层和小脑小叶中。考虑到效力、成像和稳定性，[Q1G，ΔR14]LvΙB-BODIPY FL 是研究 nAChRs 组织分布和功能的理想药理学工具。我们的研究结果不仅为将芋螺毒素开发为可视化药理学探针提供了基础，还展示了 nAChRs 在大鼠大脑中的分布。
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