The ion channels in the apical and basolateral membranes of colonic epithelial cells are essential elements in the transport functions of this organ; and it is likely that the modulation of ion channel properties plays a pivotal role in the regulation of Na absorption and secretion. Recent studies of the regulation of Na absorption in tight epithelia emphasize a role for the basolateral membrane, but other results indicate that the conductive properties of the basolateral membrane are more complex than previously thought. In colonic cells, for example, basolateral K conductance is due to at least two populations of K channels which play different roles in the life of the cells. Despite enormous advances in our understanding of the properties of ion channels in neurons and artificial systems the properties of epithelial ion channels remain, by comparison, virtually undescribed. This state of affairs is in part the result of methodological limitations which have precluded the measurement of specific ion currents across epithelial cell membranes under conditions where driving forces are well-defined. We propose to study the conductive properties of the basolateral membranes of colonic epithelial cells using three techniques: 1) measurement of macroscopic currents across basolateral membranes which have been functionally isolated with amphotericin-B or digitonin. 2) Fluctuation analysis which will permit us to estimate single-channel properties from the fluctuations in macroscopic currents and 3) Patch-clamping which will allow us to study the properties of single channels and to apply voltage-clamp techniques to single cells.
Specific aims are to 1) identify specific ion channels, 2) describe regulatory influences on channels and 3) determine the physiological significance of specific populations of ion channels with regard to colonic ion transport. We will study basolateral ion channels in the intact tissue, in isolated cells and in membrane fragments using techniques which will provide access to the intracellular as well as the extracellular environment. This approach will enable us to describe the properties of single ion channels in terms of the biophysics of ion conduction and gating and to relate these to the macroscopic properties of the intact cell layer. These studies should provide insight into the mechanisms by which ion channels interact with intracellular mediators to produce highly regulated transcellular ion transport in colonic epithelial cells.
Showing the most recent 10 out of 21 publications
