The cystic fibrosis (CF) phenotype is characterized by a regulatory defect in apical membrane Cl- permeability in secretory epithelia associated with a reduction in fluid secretion. Recent investigations on Cl- secretory epithelial cells in culture have characterized the regulation of anion channels by changes in cellular volume, increases in intracellular free Ca2+, and increases in intracellular cyclic nucleotides. It is our hypothesis that Ca2+-dependent Cl- channels (I-Cl, Ca) will provide for an alternative regulated Cl- conductance pathway to correct the transport dysfunction in CF affected cells. This pathway is not defective in CF tissue and may, in fact, be upregulated in human airway tissue. Ca2+-dependent Cl- conductances in secretory epithelium can be separated into two distinct populations: those that are directly regulated by Ca2+ and those which are inhibited by CaMKII inhibitors. In preliminary studies we have cloned the human isoform of the ClC-3 chloride channel from the human colonic cell line T84, and carried out electrophysiological studies which demonstrate that CaMKII is involved in the activation of hClC-3. In the proposed studies, we will further characterize the cloned channel and I-Cl,CaMKII by investigating its gating mechanism and pharmacological properties. (1) We will examine the identity of hClC-3 and I-Cl,CaMKII by antisense and knock-out mice to eliminate endogenous hClC-3. We will characterize the response of recombinant hClC-3 to specific CaMKII inhibitors. (2) We will determine the role of CaMKII in gating and/or translocation of the channel using established cell lines and primary culture from ClC-3 (-/-) knockout mice using whole cell and single channel analysis. (3) We will further investigate the role of CaMKII phosphorylation in the activation of hClC-3 by site-directed mutagenesis and phosphorylation assays as well as electrophysiological studies. We will evaluate the physiological role and possible interplay between phosphorylation-dependent activation of the channel by CaMKII and the inhibition of channel activation by annexin IV and IP4. (4) We will investigate an approach to augment I-Cl,CaMKII in CF cells via either a constitutively active CaMKII or peptide that blocks the interaction between hClC-3 and its specific inhibitors, such as annexin IV and IP4. The ultimate goal of this investigation is to provide candidate intracellular therapeutic targets which may be useful in ameliorating the chronic pathological condition associated with CF.

R01DK057882

2001-07-01

2006-05-31