Research Objective

1. My group is studying storage of the neurotransmitter acetylcholine (ACh) by synaptic vesicles, which are hollow storage organelles located in nerve terminals. When a nerve impulse arrives at the terminal, it causes synaptic vesicles to secrete ACh into the synaptic gap where it acts to propagate the signal to the next cell. Synaptic vesicles are used many times, so they must be refilled between nerve impulses. Refilling is mediated by a transporter called the vesicular ACh transporter (VAChT). VAChT resides in the vesicular membrane and exchanges one ACh molecule from cytoplasm for two vesicular protons that are supplied by a separate proton pump. ACh inside synaptic vesicles is 100-fold concentrated compared to ACh in the cytoplasm. VAChT is potently inhibited by a drug called vesamicol, which acts at an allosteric site.
2. We are characterizing structure-function in VAChT with the long term aim of learning how to stimulate ACh storage pharmacologically. We have developed a kinetics model that describes individual steps in the overall transport cycle. Our experiments involve expression of cloned VAChT in a mammalian cell line called PC12. The cDNA for VAChT is mutated to change potentially interesting amino acid residues, and mutated VAChT located in vesicles is isolated from PC12 cells. The preparation is characterized as to ACh and vesamicol binding affinities, Km and Vmax values for ACh transport, and the pH dependencies of all parameters. Partial identification of binding sites for ACh, vesamicol and protons has been achieved. Also, important sites of flexibility in the polypeptide backbone that control rates of conformational change during transport have been identified.
3. Our long term goal is to understand how the VAChT works and is regulated. The information might present an opportunity to increase ACh storage in and subsequent release from distressed nerve terminals, such as occurs in Alzheimer's and other diseases.

In other work, we are developing rapid and inexpensive tests for the compound gamma-hydroxybutyrate (GHB). GHB produces sedation and a trance-like state with loss of memory. Because it has little smell or taste, it can be ingested unknowingly. This combination of properties has made GHB a "date rape" drug that often is administered to victims in beverages. Current assays for GHB either require sophisticated equipment, a skilled person and a long time period to complete, or they use chemistry that is hazardous, multi-step, insensitive and very susceptible to false positives. We have developed an enzymatic test that uses cloned GHB dehydrogenase. Oxidation of GHB is coupled to the reduction of a pro-dye that yields a highly colored product in seconds.