Patch clamp studies have identified a cAMP-regulated chloride channel in both B- and T-lymphocyte cell lines (Chen, Schulman and Gardner. Science 234:657, 1989). It has current-voltage relations, kinetic behavior, and regulatory properties similar to those described for secretory epithelial cells. The regulation of the channels by adenosine 3'5'-monophosphate (cAMP)-dependent protein kinase in transformed B cells from cystic fibrosis (CF) patients is defective. Thus the CF-encoded defect in malfunctioning chloride channels in the somewhat inaccessible epithelial cells that line airways can now be studied in the more accessible lymphocyte model. This should aid biochemical, molecular, and genetic studied os the disease. In addition, identification of a primary defect in immune cells may provide new insights into the pathophysiology of CF. The overall goals of this proposal are to more fully characterize the basic biophysical properties of the lymphocyte chloride channel, to delineate the regulatory pathways of channel activation, to directly compare chloride channels in lymphocyte and the secretory epithelial cell line T84, and to determine if the channel is also present in peripheral blood lymphocytes (PBL's). Some of the biophysical properties to be addressed include relative anion permeability, voltage- and time-dependence of channel gating parameters, sensitivity to specific chloride channel blockers, and assessment of the aggregate chloride current by the whole-cell clamp technique. Regulatory studies will assess the three pathways of channel activation, including: 1) activation in excised patches by sustained depolarization; 2) activation following phosphorylation by cAMP-dependent protein kinases (either on -cell by cAMP-dependent analogs or in excised patches exposed to purified subunit of cAMP-dependent protein kinase plus ATP; 3) activation via an alternative Ca2+-dependent pathway, as indicated in on-cell patches of cells exposed to calcium ionophore A23187. The goals will be to fully characterize the three regulatory pathways including the relevant effector molecules, to compare channel gating kinetics to determine clues to structure-function relationships, and to determine if the third Ca2+-dependent pathway is preserved in CF-derived cells as preliminary studies indicate. All studies will be performed in a comparative manner in cells of the secretory colonic epithelial cell line T84 and in PBL's. It is hoped that these studies will provide insight into the general knowledge of ion channel regulation and function and furnish a valuable model for the chloride channel dysfunction associated with CF.
