Although we know that the rectifying chloride channel is defective when mutant CFTR is expressed, the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR) and other ion channels in cystic fibrosis (CF) tissues is not known. Cystic fibrosis (CF) is also characterized by increased sodium absorption. This increased sodium absorption adds to the reduced chloride secretion to further diminish normal fluid balance. If sodium hyperabsorption or normal sodium absorption can be inhibited, it may mitigate compromised fluid balance. The cellular basis of the sodium defect in CF is unknown. One possibility is increased absorption through amiloride-sensitive sodium channels. Since nucleotide-gated cation channels are abundantly expressed in lung (bronchi and bronchioles) and gut, (villus cells), two organs involved in cystic fibrosis, this channel may also contribute to hyperabsorption of sodium. If nucleotide-gated cation channels participate in normal sodium absorption or excessive sodium absorption in CF, the dichlorobenzamil inhibitory site on this channel may be a potential site for therapeutic intervention.
The Specific Aims are: 1) To evaluate whether nucleotide-gated cation channels contribute to sodium absorption in tissues involved in CF by determining a) the distribution of nucleotide-gated channels by in situ hybridization in normal seat glands, pancreatic cells and the nasal epithelium b) immunocytochemical localization of the channel in lung, intestine, pancreas and sweat glands and c) the contribution of nucleotide-gated channels to short circuit current of rat tracheal cells. 2) To evaluate the interaction between amiloride-sensitive sodium channels and nucleotide-gated cation channels and CFTR by cotransfection of nucleotide-gated channels with mutated or normal CFTR for study of the properties of channels which may be responsible for hyperabsorption of sodium. Comparisons will be made using whole-cell and single channel recordings. 3) To clone, then characterize the pharmacological and electrophysiological properties of the novel intestinal nucleotide-gated cation channel by expression of cDNA clone(s) in oocytes. To determine if dichlorobenzamil blocks the condition pore of the nucleotide-gated channel.
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