The aminoacid/ polyamine/organocation (APC) superfamily of transporters includes human LAT and CAT amino acid transporters as well the Na-K-Cl, Na-Cl and K-Cl cation-chloride cotransporters (CCCs). The CAT and LAT amino acid transporters mediate a major fraction of amino acid movement across cell membranes and across renal and intestinal epithelia. The Na-K-Cl cotransporter, Na-Cl, and K-Cl cotransporters play a central role in cellular volume and chloride homeostasis, and in neuronal cells are responsible for regulation of excitability through changes in intracellular chloride. In secretory epithelia, NKCC1 functions together with Cl channels, the Na pump, and K channels to bring about the major fraction of regulated salt secretion movement, while in the mammalian kidney another isoform, NKCC2, mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. None of these transport proteins is understood at the molecular level because we do not know the structure of any of the APC family members. The goal of this project is to obtain the first high-resolution crystal structure of a prokaryotic APC superfamily member, and to understand the function of the transporter in the context of the structure. We have chosen prokaryotic APCs because we can select among a large number of potential candidates, and because we can express these proteins at high levels in bacteria. Strong alignment assures that we will be able to apply the structural information to high-resolution predictions for the structure of the eukaryotic transporters. This high-payoff project is paradigm-shifting in that it will open the door to a broad field of investigation in amino acid transporters and cation-chloride cotransporters in work in which structural information is used to inform mechanistic studies, including studies of regulation and drug interactions.
The specific aims are 1) to clone a diverse set of prokaryotic cationic amino acid transporters and examine their ability to be produced in high yield and with high stability, 2) to obtain the high resolution crystal structure of an APC transporter, and 3) to determine the function of selected prokaryotic transporters, relating the function to the emerging structure. Diseases and disease conditions including hypertension, cerebral edema, polycystic kidney disease, secretory diarrhea, cystic fibrosis, and some diseases of the nervous system involve defects or over activity of the cellular machinery that is responsible for salt and amino acid movements across cell membranes. This research is directed to understanding one part of that cellular machinery, a set of related proteins called cation- chloride cotransporters and amino acid transporters that are responsible for directly handling coordinated sodium, potassium and chloride movements, or amino acid movements. By understanding the molecular structure of these proteins, we will be able to understand the mechanics of their action, and the mechanism of their regulation, thus being better able to design diagnostic and therapeutic agents and treat the disease states.
