# Structures and Pharmacology of Cation-Chloride Cotransporters

> **NIH NIH R01** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2021 · $335,500

## Abstract

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.

## Key facts

- **NIH application ID:** 10367176
- **Project number:** 1R01DK128592-01A1
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** Erhu Cao
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $335,500
- **Award type:** 1
- **Project period:** 2021-09-20 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10367176

## Citation

> US National Institutes of Health, RePORTER application 10367176, Structures and Pharmacology of Cation-Chloride Cotransporters (1R01DK128592-01A1). Retrieved via AI Analytics 2026-06-10 from https://api.ai-analytics.org/grant/nih/10367176. Licensed CC0.

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