PILOT--POTASSIUM CHANNEL PROPERTIES OF AIRWAY CELLS

囊性纤维化的特征是对Na+的高吸收，以及对cAMP介导的激动剂的CI分泌反应减弱或消失。在这些观察的基础上，探索了三种不同的药理方法。这些包括抑制Na+转运，直接药物激动剂激活CFTR，以及可能绕过原发CF缺陷的替代电导的激活。根据根尖和基底侧膜之间的电耦合需要维持CI分泌的Na+吸收，调节基底侧膜的K+电导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)通道已被广泛克隆和研究。然而，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还没有在单通道水平上得到鉴定，也没有从分子水平上得到鉴定。对这些钾通道的了解对于确定离子在人体呼吸道中的传输至关重要。我们的初步研究表明，这些K+通道代表了呼吸道细胞的静息电导。我们建议使用全细胞和单通道膜片钳技术来研究原代培养的人支气管上皮细胞(HBE)和浆液性细胞系CALU-3中的钾通道。我们假设最近克隆的两个孔域K+通道家族负责这些电导。当异源表达时，这些通道被证明是结构性活跃的，并被认为是细胞中的背景或静止的K+电导。然后，我们将使用基于RT-PCR的方法来扩增HBE和CALU-3细胞的两个孔域类别的钾通道，我们假设这两个通道对这些离子运输过程至关重要。一旦鉴定出这些克隆，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.