Electrolyte transport by gastrointestinal and renal epithelial cells is mediated by an array of coupled transporters, ATPases and ion channels. Absorption generally occurs via coupled solute entry across the lumenal border driven by an inwardly directed Na+ gradient established by the (Na+ + K+)-ATPase on the serosal surface. Secretion of Cl- into the lumenal compartment is mediated by specific ion channels and provides the driving force for the osmotic movement of water. It is likely that similar elements mediate the volume regulatory responses to osmotic perturbations. Although the stimuli for these responses are well- defined, the modes of activation are only partly understood. Recently we have isolated a subcellular membrane fraction that is enriched in a single type of Cl- channel. The membrane has a characteristic protein composition thus antibodies that are specific for these membrane proteins allow their identification and localization to specific cell types. Although we do not know the function of these channels it is interesting to note that all of the cells and tissues thus far demonstrated to have these antigens are involved in Cl- secretion or absorption. In particular, T84, cells, a model secretory cell derived from a human colonic tumor, possess a comparable membrane fraction exhibiting Cl- channel activity. The hypothesis to be tested is that there are two levels of control of ion transport in these tissues: (1) direct activation of channels in the plasma membrane and (2) a change in the overall number of transporters in the plasma membrane by recruitment or cycling through a subcellular pool. The hypothesis will be tested by focusing on T84 cell Cl- transport. Antibodies and biochemical probes to specific membrane proteins will be used to examine membrane turnover in confluent, functionally intact T84 cell monolayers grown on permeable supports. The antibodies will be generated by purification of membranes and proteins from more readily accessible bovine tissue. The channel protein(s) will be purified using reconstitution to estimate purity. Relevant proteins, identified as having a functional role in reconstitution experiments, will be partially sequenced to provide information to synthesize oligonucleotide primers. Purified mRNA from bovine trachea will be used as template in the polymerase chain reaction to generate a cDNA specific for channel protein. The cDNA will provide true sequence information for synthesis of specific oligonucleotide probes to screen cDNA expression libraries.
