Structures and Pharmacology of Cation-Chloride Cotransporters - Project Summary Human cation-chloride cotransporters (CCCs) consist of three Na+-dependent Na+-(K+)-Cl- (NCC and NKCC1- 2) and four Na+-independent K+-Cl− (KCC1-4) transporters. CCCs drive the symport of Cl- with Na+ and/or K+ across membrane and play pivotal roles in cell volume regulation, trans-epithelia ion movement, and regulation of intracellular Cl- concentration ([Cl-]i) and neuronal excitability. CCCs are profoundly regulated by the opposing actions of WNKs-SPAK kinases and phosphatases. Intriguingly, N(K)CC and KCCs exhibit inverse phosphoreg- ulatory mechanism, with the former activated and the latter inhibited by phosphorylation. In the kidneys, NKCC2 and NCC reabsorb ions from the forming urine, maintaining electrolyte balance, blood volume, and pressure. Mutations in NCC, NKCC2, or WNKs and their upstream E3 ubiquitin ligase degraders lead to hypotensive dis- orders (Gitelman and Batter syndromes) or Familial Hyperkalemic Hypertension. CCCs have been prolific targets for FDA-approved drugs. Loop and thiazide diuretics inhibit NKCC2- and NCC-mediated renal salt retention, respectively, and have been cornerstones in the treatment of hypertension and edema since the 1950s. How- ever, loop diuretics also inhibit NKCC1 and KCCs, and this off-target antagonism can cause temporary or even permanent hearing loss. In the brain, NKCC1 and KCC2/3 are the major Cl- loader and extruders, respectively. Their balanced actions set [Cl-]i away from electrochemical equilibrium, allowing inhibitory neurotransmitters to evoke either inward depolarizing or outward hyperpolarizing Cl- currents via ligand-gated anion channels. Muta- tions in KCC2/3 cause seizures, epilepsy, and other brain disorders, likely due to an imbalance in excitatory versus inhibitory synaptic transmission resulting from deranged [Cl-]i. Pharmacological modulation of NKCC1 and KCC2/3 transport activities is therefore a promising strategy to restore synaptic inhibition and treat numerous brain disorders. Finally, CCCs are expected to be regulated by and signal through associated protein partners, but such decisive CCC regulators and effectors remain to be discovered. Here we propose to determine struc- tures of CCCs using cryo-EM and perform associated biochemical and functional studies to elucidate: i) how diverse chemical classes of loop diuretics bind to and form chemical bonds with the therapeutic target NKCC2 and the off-target KCC3; ii) why (de)phosphorylation inversely regulates NKCC2 versus KCCs; and iii) how KCC3 is regulated by and signals through a novel partner that we discovered via proteomics and validated by deter- mining its co-structure with KCC3. In parallel, we have developed robust ion flux assays to accelerate CCC structure-function relationship studies. In the longer term, these functional assays could evolve into high-through- put screening platforms for the discovery of compounds and biologics. Such pharmacological agents could be used to probe the structures and functions of CCCs and serve as drug leads for treating hypertension, edema, and brain disorders. Overall, our multidisciplinary approaches will help decipher the inner workings of CCCs and facilitate the rational targeting of these transporters for the treatment of numerous CCC-associated disorders.
