PILOT--POTASSIUM CHANNEL PROPERTIES OF AIRWAY CELLS

囊性纤维化（CF）的特点是Na+的高度吸收以及CI对camp介导的激动剂的分泌反应减弱或缺失。基于三项观察，探索了三种不同的药理学方法。这些包括Na+转运抑制剂，CFTR的直接药理激动剂激活和可能绕过原发CF缺陷的替代电导的激活。根据维持Na+吸收和ci分泌所需的根尖膜和基底外侧膜之间的电偶联，基底外侧膜的K+电导调节是维持这些离子传输过程的必要条件。参与这些过程的Na+ (EnaC)和Cl-(CFTR)通道已被克隆和广泛研究。然而，对于维持Na+吸收和ci分泌的电化学驱动力至关重要的钾通道尚未在单通道水平上确定，也未在分子水平上确定。了解这些钾离子通道对于确定离子在人体气道中的转运至关重要。我们的初步研究表明，这些K+通道代表了气道细胞的静息电导。我们建议使用全细胞和单通道膜片钳技术来表征人支气管上皮细胞（HBE）和浆液细胞系calu-3的三种钾通道。我们假设最近克隆的两个孔域K+通道家族负责这些电导。这些通道已被证明在异源表达时具有组成性活性，并被认为在细胞中呈现背景或静止K+电导。然后，我们将使用基于RT-PCR的方法从HBE和calu-3细胞中扩增两种孔域类钾通道，我们假设这两种通道对这些离子运输过程至关重要。一旦这些克隆被确定，我们将使用反义寡核苷酸选择性地敲除这些电导，并确定它们对HBE和calu-3细胞中离子运输的影响。

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.