The epithelial sodium channel of rabbit colon: Moleculare structure, function and regulation

The apical (outward-facing) cell membranes of many epithelia contain highly-selective sodium channels that are stimulated by the hormone aldosterone and inhibited by the diuretic amiloride. Such channels are present in the distal colon, the collecting ducts of the kidneys, and the lung. They mediate the first step of active sodium absorption and play a major role in the maintenance of electrolyte and water homeostasis as well as blood pressure. The elucidation of the mechanism and regulation of epithelial sodium absorption is therefore of basic physiological, pathophysiological and clinical relevance. It is the purpose of the proposed research project to study the function, regulation, and structure of the epithelial sodium channel from rabbit colon at the single-channel, i.e. at the molecular level. Single-channel activity will be recorded by reconstituting apical membrane vesicles of rabbit colon epithelium into artificial planar lipid bilayers. Using this technique, both sides of the channel are easily accessible, hence potential extra- and intracellular regulators can be tested directly. The regulators to be examined include sodium, calcium, pH, G-proteins, phosphorylation and dephosphorylation reactions, and the phospholipid composition of the bilayers. The chemical entities of the channel essential for conductance and gating will be characterized using group-selective reagents. The structure of the epithelial sodium channel from rabbit will be defined by cloning this channel. By comparing the functional properties of the cloned epithelial sodium channel expressed in frog eggs (Xenopus laevis oocytes) with those of the native channel, evidence can be derived whether the native channel is equipped with regulatory mechanisms that are absent in the cloned channel. Using Northern-blot analysis, it is intended to study the regu- lation of the channel subunits by steroid hormones in different organs. Expression of trun-cated or otherwise altered channel subunits in Xenopus oocytes may help to understand the functional role of different regions of the channel proteins.