Chloride transport plays a major role in the secretion of salts and fluids and in response to osmotic challenge. Fluid secretion in epithelial cells is modulated by the second messenger cAMP. The regulation of cAMP- dependent Cl- channels on the apical membrane of secretory epithelia contributes to the rate determining step for fluid secretion in these cells. This cAMP-dependent cl- transport is disrupted in Cystic Fibrosis (CF), a human genetic disease. Several cAMP-dependent cl- channels, whose activity may underlie macroscopic Cl- conductance, have been described in epithelial cells, under different experimental conditions. Two channels of primary interest are a large (40-45 pS), outwardly rectifying Cl- channel and a small (8-10 pS), linear Cl- channel. The activity of both of these channels is increased by cAMP-dependent mechanisms. The relationship between cAMP- dependent single channel and macroscopic Cl- current will be addressed by asking: What single Cl- channel activity can be identified in the presence of cAMP? Can the properties of one specific cAMP-dependent single channel activity account for the properties of the macroscopic current? These questions will be addressed as simultaneously recording cAMP- dependent single Cl- channel activity and macroscopic Cl- current. Once the ion channel(s) involved have been identified, their selectivity, permeation and kinetic properties, and pharmacological profile of their ion channel(s) will be characterized. Possible interactions between cAMP- and Ca++-dependent second messenger systems on single channel and macroscopic Cl- current activity will also be investigated. In the broad prospective, this project will address the physiological role of Cl- channels by elucidating the basic properties and regulatory mechanisms of Cl- channels at the single channel and macroscopic levels. The intimate knowledge of these properties will help to bridge the gaps between the molecular and cellular levels of secretory physiology. Thus, by identifying the underlying molecular events of cAMP-dependent Cl- transport in epithelial cells, we will be able to further the understanding of the role of these ion channel proteins play in fluid secretion. This will also provide valuable insight into the mechanistic basis of abnormal fluid secretion in Cystic Fibrosis.
