Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes

Abstract

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: (a) carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; (b) carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulate gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: (a) if sufficiently bulky, they inhibit transport; (b) they bind outside the substrate site (though adjacent to it), (c) they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel; the choline carrier binds monoalkyl-substituted substrate analogs in the outer gated channel and N-ethylmaleimide and a hydrogen ion in the inner gated channel (the hydrogen ion adding to an ionizing group of pKa 6.8). Inhibitors of this kind may be used to provide evidence on the nature of the complementary peptide surfaces forming the channel walls, as well as evidence on the mechanism of gating.