Regulation of function of the solute carrier sodium-coupled choline transporter CHT

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The overall objective of this research program is to develop greater understanding of mechanisms involved in regulation of secondary-active sodium-coupled solute transporters such as the high-affinity choline transporter CHT that is expressed predominantly in cholinergic neurons. Cholinergic neurons are found in organisms from invertebrates to humans where they use the neurochemical transmitter acetylcholine (ACh) to communicate information to a variety of target organs and cells. In mammals, they transmit critical information in both peripheral and central nervous systems, controlling diverse functions from neuromuscular contraction and hormone secretion to sleep, attention and higher-order cognitive functions. ACh is synthesized by choline acetyltransferase from choline that is taken up into cholinergic neurons by CHT, but little is known about the dynamic events that regulate and coordinate CHT function. This grant proposal takes studies on CHT in exciting new directions to identify and characterize key events regulating CHT. We propose a model where a recycling pool of CHT proteins interacting with cellular proteins are regulated in cholinergic terminals during neuron activity to drive ACh synthesis; these interacting proteins are functionally or physically coupled to position CHT in the cell and facilitate efficient ACh synthesis. Neither the mechanisms by which CHT is trafficked within the cell or forms functional complexes with other proteins, nor the physiological signals that regulate these processes, are known. The aims of this research program are to identify and define cellular-molecular mechanisms involved in acute regulation of CHT proteins related to their trafficking and subcellular distribution [Aim #1] and interaction with other cellular proteins [Aim #2]. In relation to their subcellular trafficking and processing, some transporters form functional homo-dimers or oligomers, but this is unknown for CHT. Key points to be identified are where dimerization occurs in the cell, the function and longevity of complexes, signals that stimulate their formation, and the role in cellular communication. Based on our previous studies, we have identified potential CHT interacting proteins that may regulate its trafficking between subcellular compartments and the cell surface where it expresses its functional solute transport activity. LC2 and NECAP are two such candidate proteins. In our approach to these investigations, we being together our experience in cholinergic neuron biology and expertise using a diverse range of cell and molecular biology methods and advanced imaging technology in live cells to solve these long-standing questions. Importantly, when taken together, much of the information gained in these studies will also have more general relevance to the regulation of a range of plasma membrane proteins that mediate solute transport into cells.