PILOT--POTASSIUM CHANNEL PROPERTIES OF AIRWAY CELLS

先导--气道细胞的钾离子通道特性

• 批准号: 6654126
• 负责人: DANIEL C DEVOR
• 金额: $ 12.41万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6654126
• 关键词: antisense nucleic acid; cell line; cystic fibrosis; ion transport; membrane permeability; membrane potentials; molecular cloning; polymerase chain reaction; potassium channel; protein structure function; respiratory epithelium; tissue /cell culture; voltage /patch clamp

Cystic fibrosis (CF) is characterized by both a hyper-absorption of Na+ as well as a diminished or absent CI secretory response to cAMP-mediated agonists. Based on thee observations, three distinct pharmacological approaches have been explored. These include inhibitor of Na+ transport, activation of CFTR by direct pharmacological agonists and activation of alternative conductances that may circumvent the primary CF defect. Based upon the requirement for electrical coupling between apical and basolateral membranes to sustain either Na+ absorption of CI-secretion regulation of a basolateral membrane K+ conductance is a requisite to the maintenance of these ion transport processes. The Na+ (EnaC) and Cl-(CFTR) channels involved in these processes have been cloned and studied extensively. However, the potassium channels critical to the maintenance of the electrochemical driving force for Na+ absorption and CI-secretion have not been identified at the single channel level nor he they been molecularly identified. An understanding of these potassium channels is critical to defining ion transport across the human airway. Our preliminary studies suggest that these K+ channels represent the resting conductances of the airway cells. We propose to characterize thee potassium channels in both primary cultures of human bronchial epithelial (HBE) and the serous cell line, calu-3 using whole-cell and single-channel patch-clamp techniques. We hypothesize that the recently cloned family of two pore domain K+ channels re responsible for these conductances. These channels have been shown to be constitutively active when heterologously expressed and are believed to present background or resting K+ conductances in cells. We will then use a RT-PCR based approach to amplify the two pore-domain class of potassium channels from HBE and calu-3 cells which we hypothesize are critical to these ion transport processes. Once these clones are identified we will use antisense oligonucleotides to selectively knockout these conductances and determine their effects on ion transport across HBE and calu-3 cells.

囊性纤维化（CF）的特征是Na+的高吸收以及对CAMC介导的激动剂的CI分泌反应减少或不存在。根据您的观察，已经探索了三种不同的药理学方法。这些包括Na+转运的抑制剂，通过直接药理激动剂激活CFTR以及可能绕过主要CF缺陷的替代电导。基于对顶端和基底外侧膜之间的电耦合的要求，以维持基底外侧膜K+电导的CI分泌调节，这是维持这些离子运输过程的必要条件。这些过程中涉及的Na+（ENAC）和Cl-（CFTR）通道已被广泛克隆和研究。然而，尚未在单个通道水平上鉴定出对维持Na+吸收和CI分泌的电化学驱动力至关重要的钾通道，也没有在分子上鉴定出来。对这些钾通道的理解对于定义跨人类气道的离子运输至关重要。我们的初步研究表明，这些K+通道代表气道细胞的静息电导。我们建议在人支气管上皮（HBE）和浆液细胞系的主要培养物中表征您的钾通道，即使用全电池和单渠道补丁钳技术CALU-3。我们假设最近克隆的两个孔域K+通道对这些电导负责。当异源表达时，这些通道已被证明是组成型活性，并且据信在细胞中呈现背景或静止的K+电导。然后，我们将使用基于RT-PCR的方法来放大来自HBE和Calu-3细胞的两个孔隙域钾通道，这对于这些离子传输过程至关重要。一旦确定了这些克隆，我们将使用反义寡核苷酸选择性敲除这些电导，并确定它们对跨HBE和Calu-3细胞的离子转运的影响。