In the physiology of transport through biological membranes the mechanisms have been classified as either carrier-type or channel-type by their kinetic characteristics, for example by their relative tracer and net permeabilities. The corresponding transport proteins were thought to be structurally different in that a carrier possessed a more or less mobile substrate binding site and a channel possessed an essentially stable structure that remained immobile during substrate translocation. The proposed study is an experimental test of a new hypothesis unifying carrier-and channel-mediated substrate transport across biological membranes. In this hypothesis a biological carrier is viewed as a specialized channel. This channel can exist in two basic conformations which differ in their internal potential energy profiles. The carrier-type kinetics arise from conformational changes between these two states which alternately lower and restore the barriers on the two ends of the channel, thus alternately exposing the central substrate binding site to the two sides of the membrane. The difference between this mechanism and previous carrier-type models is that two modes of net transport are predicted: by slippage, the conformational change of the empty channel; and by tunneling, the movement through the channel without a conformational change. This hypothesis will be quantitatively tested with the anion transporter of the human erythrocyte. The objectives are to 1) dissect and characterize the different components of anion net flux; 2) evaluate the hypothesis by testing several specific predictions; and 3) determine the ligand binding properties of the anion binding site from the kinetics of the tunneling component of net flux. Experiments will be performed with intact cells and resealed ghosts. I will measure net efflux and influx of chloride and sulfate at different intracellular and extracellular concentrations and pH. Since all biological carriers and active transporters may be specialized channels, this study should provide important general insights into their function. This study of the anion transporter will be the first test of the hypothesis unifying biological transport mechanisms.
