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首页> 外文学位 >Pharmacological and genetic manipulation of small conductance calcium-activated potassium channels in adrenal chromaffin cells.
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Pharmacological and genetic manipulation of small conductance calcium-activated potassium channels in adrenal chromaffin cells.

机译：肾上腺嗜铬细胞中小电导钙激活钾通道的药理和遗传操作。

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摘要

Since their molecular identification, small conductance calcium-activated potassium channel have emerged as important regulators of membrane excitability in central nervous system and in neuronal tissues. Three isoforms of the SK channel exist: SK1--SK3. These channels are subjected to multi-faceted regulation from the transcriptional to post-translational levels. The members of the SK channel family along with IK channel share a great deal of sequence conservation, and all of them use calmodulin as a beta subunit that confers Ca2+ sensing activity. One consequence of multi-level regulation of SK channels is control of cellular localization.; We discovered a novel genetic tool for silencing SK and IK channels, SK3-1C. This naturally occurring transcript has been extensively characterized, and the domain responsible for the ability of SK3-1C to inhibit native currents in the adrenal chromaffin cell carcinoma (pheochromacytoma) cell line, PC12, has been identified. The extensive conservation in the dominant inhibitory sequence, or DIS, indicates that SK3-1C can silence other members of the SK channel family. Whole cell patch clamp experiments revealed that SK3-1C is able to silence the entire SK channel family as well as the IK channel. Immunocytochemistry using and anti-SK3 antibody and confocal microscopy has determined SK3 channel localization in PC12 cells transfected with SK3-1C is dramatically altered.; The presence of SK3 subunits in PC12 cells indicated that SK channels may play an important role in normal adrenal tissue. Immunohistological and immunocytological experiments indicate that SK3 is the predominant channel found in the adrenal medulla, while SK2 appears to predominate in the adrenal cortex. SK channels also appear to be expressed in adrenomedullary chromaffin cells (ACCs) that also express the enzyme phenylethanolamine-N-methyl transferase (PNMT), which is a marker for epinephrine secreting cells. SK channels tend to reduce the excitability of ACCs, as reduction in activity of SK channels using inhibitor compounds increases the action potential firing rate and the opener NS309 slows down firing. ACCs from mice overexpressing SK2 and SK3 exhibit reduced excitability comparable to NS309 treatment, though the effects of overexpressing SK3 are much more severe. Ca2+ influx induced by slight increases in external K+ concentrations were also enhanced by SK blockade and reduced by NS309. Lastly, preliminary data from analysis of catecholamines in cell culture medium from ACCs treated with high K+ and SK channel pharmacological agents is encouraging as a potential method to analyze catecholamine secretion without the difficulties in controlling experiments done in live animals. Throughout the study of SK channels and the adrenals, several future directions for study of SKs in this tissue have emerged.
机译：由于其分子鉴定，小电导钙激活钾通道已成为中枢神经系统和神经元组织中膜兴奋性的重要调节剂。 SK通道存在三种同工型：SK1-SK3。这些通道受到从转录到翻译后水平的多方面调节。 SK通道家族和IK通道的成员共享大量的序列保守性，并且所有人都使用钙调蛋白作为赋予Ca2 +感应活性的β亚基。 SK信道的多级调节的结果之一是对细胞定位的控制。我们发现了一种用于沉默SK和IK通道的新型遗传工具SK3-1C。该天然存在的转录本已得到广泛表征，并且已经确定了负责SK3-1C抑制肾上腺嗜铬细胞癌（嗜铬细胞瘤）细胞系PC12中的天然电流能力的结构域。显性抑制序列或DIS的广泛保守性表明SK3-1C可以沉默SK通道家族的其他成员。全细胞膜片钳实验表明，SK3-1C能够使整个SK通道家族和IK通道静音。使用抗SK3抗体和共聚焦显微镜进行的免疫细胞化学测定已确定，用SK3-1C转染的PC12细胞中的SK3通道定位发生了巨大变化。 PC12细胞中SK3亚基的存在表明SK通道可能在正常的肾上腺组织中发挥重要作用。免疫组织学和免疫细胞学实验表明，SK3是在肾上腺髓质中发现的主要通道，而SK2在肾上腺皮质中似乎占主导。 SK通道似乎也在肾上腺髓质嗜铬细胞（ACC）中表达，肾上腺嗜铬细胞也表达苯乙醇胺-N-甲基转移酶（PNMT），这是肾上腺素分泌细胞的标志物。 SK通道往往会降低ACC的兴奋性，因为使用抑制剂化合物降低SK通道的活性会增加动作电位的发射速度，而开孔剂NS309会降低发射速度。与NS309治疗相比，过表达SK2和SK3的小鼠的ACC表现出降低的兴奋性，尽管过表达SK3的作用更为严重。外部K +浓度略有增加引起的Ca2 +内流也被SK阻滞增强，而被NS309抑制。最后，来自高浓度K +和SK通道药理剂处理过的ACC细胞培养基中儿茶酚胺分析的初步数据令人鼓舞，它是分析儿茶酚胺分泌的一种潜在方法，而没有控制活体动物实验的困难。在整个SK通道和肾上腺的研究中，已经出现了在该组织中研究SK的几个未来方向。

著录项

  • 作者

    Kolski-Andreaco, Aaron Asher.;

  • 作者单位

    University of California, Irvine.;

  • 授予单位 University of California, Irvine.;
  • 学科 Biology Physiology.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 144 p.
  • 总页数 144
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物物理学;
  • 关键词

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