PILOT--POTASSIUM CHANNEL PROPERTIES OF AIRWAY CELLS

囊性纤维化（CF）的特征在于Na+的过度吸收以及对cAMP介导的激动剂的Cl分泌应答减少或不存在。基于这些观察，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、通过直接药理学激动剂激活CFTR和激活可绕过原发性CF缺陷的替代传导。基于顶侧膜和基底侧膜之间的电耦合以维持Na+吸收或CI分泌的需要，基底侧膜K+电导的调节是维持这些离子转运过程的必要条件。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Cl-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鉴定，也未在分子水平上鉴定。了解这些钾离子通道对于确定离子通过人体气道的转运至关重要。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细胞（HBE）和浆液性细胞系calu-3的原代培养物中，使用全细胞和单通道膜片钳技术来表征三个钾通道。我们推测，最近克隆的家庭的两个孔域K+通道重新负责这些电导。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息K+电导。然后，我们将使用基于RT-PCR的方法来扩增来自HBE和calu-3细胞的两个孔结构域类的钾通道，我们假设这两个孔结构域类对这些离子转运过程是至关重要的。一旦这些克隆被鉴定，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细胞中离子转运的影响。

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.