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.
Showing the most recent 10 out of 25 publications
