Project Summary Human secondary active cation-chloride cotransporters (CCCs) fall into two classes: three Na+-dependent Na+- (K+)-Cl- (NCC and NKCC1-2) and four Na+-independent K+-Cl− (KCC1-4) transporters. CCCs catalyze electroneutral symport of Cl- with Na+ and/or K+ across membrane and are fundamental in cell volume regulation, trans-epithelia ion movement, regulation of intracellular [Cl-]i and neuronal excitability. In the nervous system, NKCC1 and KCC2 are the major Cl- loader and extruder, respectively, and their opposing actions move [Cl-]i away from electrochemical equilibrium so that inhibitory neurotransmitters can evoke either inward depolarizing or outward hyperpolarizing Cl- currents via pentameric ligand-gated ion channels. Mutations in KCC2 or KCC3 cause seizure, epilepsy, and other brain disorders possibly owing to an imbalance in excitatory versus inhibitory synaptic transmission. Pharmacological tuning of NKCC1 and KCCs transport activities thus represents a promising therapeutic strategy to restore synaptic inhibition for the treatment of brain disorders. In the kidneys, NKCC2 and NCC reabsorb ions from the forming urine, balancing electrolytes and blood pressure. CCCs are regulated by the WNKs-SPAK kinase cascade in response to hormone stimulation and cell volume perturbations, with N(K)CC activated and KCCs inhibited by phosphorylation. Mutations in NKCC2, NCC, or WNKs and their upstream E3 ubiquitin ligase regulators lead to hypotensive Gitelman's and Batter's syndromes or hypertensive Gordon's disease. Loop and thiazide diuretics antagonize NKCC2 and NCC, respectively, and are widely prescribed for the treatment of hypertension and edema. Building on our success in determining structures of both NKCC1 and KCC transporters in multiple states, we now propose to determine a series of new CCC structures using single-particle cryo-EM and to perform complementary biochemical and functional studies to elucidate: 1) how CCCs alternate between different transport states to shuttle ions across membranes, 2) how diuretic drugs interact with and inhibit CCCs, and 3) how (de)phosphorylation regulates CCC ion transport pathways. In parallel, we will also develop and apply cell- and liposome-based flux assays that will accelerate our CCC functional studies and, ultimately, could support high throughput screening platforms for rapid discovery of small molecule pharmacological tools to dissect CCC structures/functions and provide drug leads to treat hypertension, edema, and brain disorders. Our overarching goals are to combine structural, functional, and pharmacological approaches to understand the inner-workings of CCCs and to facilitate rational targeting of these transporters for the treatment of numerous human diseases.