Sodium crosses """"""""tight"""""""" epithelia in two steps; entry into the cell occurs across the apical membrane through a Na channel that is specifically blocked by the diuretic amiloride. We have synthesized a high affinity radioactive derivative of amiloride and used it to study the binding properties of channel-containing membranes. Further, we have produced photoactive amiloride analogs that can be specifically incorporated into these membranes. We have also raised an antibody to amiloride that can precipitate the amiloride-channel complex. Using these reagents we plan to study the characteristics of the sodium channel. We will also identify the protein which binds to amiloride. Solubilization, purification and reconstitution of the protein will be the major aim of this proposal. We will also raise antibodies to the channel and use them for further purification and for study of the synthesis and regulation of channel function.
