Neurotransmitters are chemicals which allow nerves to communicate with other nerves and organelles. Adrenaline (epinephrine) is a catecholamine type of neurotransmitter stored in the adrenal gland and released in times of stress. The adrenaline is stored inside chromaffin cells in small vesicles known as chromaffin granules. The concentration of free adrenaline in the cytosol of the cell is low (micromolar range). In contrast, the inside of the chromaffin cell is packed with adrenaline molecules at concentrations as high as 0.5 M. When the nerve is stimulated, the granules migrate to the cell membrane, fuse with the membrane and release contents into the bloodstream. The long range objective of this project is to determine the mechanism by which adrenaline is transported into these chromaffin granules. What is known so far is that a transporter molecule located in the membrane of the chromaffin granule acts to shuttle adrenaline across the chromaffin granule membrane against a high concentration gradient. Not much is known about this molecule at all, except that it needs energy to function. This energy is provided by a Mg-ATPase enzyme. The Mg-ATPase enzyme uses the energy produced from the hydrolysis of ATP to shuttle protons to the inside of the granule. These protons act to produce a pH gradient between the inside of the granule and the cytosol. In addition, the protons act to produce a positive charge on the inside of the granule resulting in a membrane potential. The driving force for the transport process appears to be both the membrane potential and the pH gradient. Major questions to be addressed are: What are the characteristics of the transporter molecule? How does this membrane potential and pH gradient act to drive the transporter? Is this transporter regulated directly by some chemical or is the synthesis of the transporter regulated? How do the characteristics of this transporter compare to those of other transporter molecules transporting adrenaline, other biogenic amines or molecules in general? Biogenic amines (neurotransmitters similar in structure to adrenaline) are stored in vesicles in the brain, and are transported into platelets; the biogenic amine histamine is stored in mast cells. Is the transporter in these vesicles different from that in the chromaffin granule? Knowledge of transporter molecules in general is limited. Studies on the adrenaline transporter will provide added insight into the general methods by which cells transport chemicals across biological membranes. One of the first steps necessary for studying the transporter is to purify the transporter and to identify the gene which codes for the transporter. The studies proposed in this research project are initial steps needed for the purification of the transporter and identification of the gene coding for the transporter.
