Chromaffin granules, the catecholamine storage vesicles of the adrenal meldulla, are a good experimental model for neurotransmitter and hormone storage vesicles of all types. Moreover, chromaffin granules have become an important system for membrane transport studies because they exhibit the H+ linked transport typical of most (and perhaps all) organelles. The granules take up catecholamines via a proton-linked transport system in which one protonated amine is exchanged for two H+. The translocator mediating this exchange is a membrane protein inhibited by the antihypertensive drug reserpine. This project will address the following questions about the catecholamine translocator in bovine chromaffin granules: 1) Does the translocator transport the protonated or the unprotonated form of the catecholamine? 2) Are Km values for efflux higher than those for influx? 3) Is transmembrane movement the ratelimiting step in transport making it valid to apply the quasi-equilibrium assumption in analyzing the kinetics of transport? 4) What are the pK values and amine binding constants of the amine/H+ translocator? To pursue these questions, two useful experimental techniques will be developed. First, a preliminary finding that reserpic acid inhibits catecholamine transport from the outside but not from the inside will be confirmed. Reserpic acid can then be used as a probe of the orientation of the transloctor. Second, a procedure for preparing inside-out membrane vesicles will be developed. This preparation will facilitate the study of the kinetics of catecholamine efflux and will also be useful in many other studies of the chromaffin-granule membrane. Using these technical tools, the kinetics of catecholamine influx and efflux will be analyzed. These data will be used to test the validity of kinetics models based on the quasi-equilibrium assumption. The objectives will be to see which models can be discount, which should be pursued and what constraints can be placed on the binding constants and pK values. Some related projects will also be pursued. First, reserpic acid affinity chromatography will be used to purify the transloctor. Second, reserpic acid will be shown to inhibit catecholamine efflux as well as influx, as expected of a competitive inhibitor. Inhibition of catecholamine efflux by the parent compound, reserpine, has never been clearly demonstrated partly because of complications caused by that drug's hydrophobicity.
