Structure of the human cation–chloride cotransport KCC1 in an outward-open state

Synaptic inhibition plays a crucial role in regulating neuronal excitability, which is the foundation of nervous system function. This inhibition is largely mediated by the neurotransmitters GABA and glycine that activate Cl--permeable ion channels, which means that the strength of inhibition depends on the Cl- gradient across the membrane. In neurons, the Cl- gradient is primarily mediated by two secondarily active cation-chloride cotransporters (CCCs), NKCC1 and KCC2. CCC-mediated regulation of the neuronal Cl- gradient is critical for healthy brain function, as dysregulation of CCCs has emerged as a key mechanism underlying neurological disorders including epilepsy, neuropathic pain, and autism spectrum disorder. This review begins with an overview of neuronal chloride transporters before explaining the dependent relationship between these CCCs, Cl- regulation, and inhibitory synaptic transmission. We then discuss the evidence for how CCCs can be regulated, including by activity and their protein interactions, which underlie inhibitory synaptic plasticity. For readers who may be interested in conducting experiments on CCCs and neuronal excitability, we have included a section on techniques for estimating and recording intracellular Cl-, including their advantages and limitations. Although the focus of this review is on neurons, we also examine how Cl- is regulated in glial cells, which in turn regulate neuronal excitability through the tight relationship between this nonneuronal cell type and synapses. Finally, we discuss the relatively extensive and growing literature on how CCC-mediated neuronal excitability contributes to neurological disorders.

Path Searching Towards the Symmetric Inward Open Structure of LeuT

The role of SLC12A family of cation-chloride cotransporters and drug discovery methodologies

Loop and thiazide diuretics have been cornerstones of clinical management of hypertension and fluid overload conditions for more than five decades. The hunt for their molecular targets led to the discovery of cation-chloride cotransporters (CCCs) that catalyze electroneutral movement of Cl- together with Na+ and/or K+. CCCs consist of two 1 Na+-1 K+-2 Cl- (NKCC1-2), one 1 Na+-1 Cl- (NCC), and four 1 K+-1 Cl- (KCC1-4) transporters in human. CCCs are fundamental in trans-epithelia ion secretion and absorption, homeostasis of intracellular Cl- concentration and cell volume, and regulation of neuronal excitability. Malfunction of NKCC2 and NCC leads to abnormal salt and water retention in the kidney and, consequently, imbalance in electrolytes and blood pressure. Mutations in KCC2 and KCC3 are associated with brain disorders due to impairments in regulation of excitability and possibly cell volume of neurons. A recent surge of structures of CCCs have defined their dimeric architecture, their ion binding sites, their conformational changes associated with ion translocation, and the mechanisms of action of loop diuretics and small molecule inhibitors. These breakthroughs now set the stage to expand CCC pharmacology beyond loop and thiazide diuretics, developing the next generation of diuretics with improved potency and specificity. Beyond drugging renal-specific CCCs, brain-penetrable therapeutics are sorely needed to target CCCs in the nervous system for the treatment of neurological disorders and psychiatric conditions.

The cation chloride cotransporter (CCCs) family comprises of four subfamilies-K(+)-Cl(-) cotransporters (KCCs), Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), and Na(+)-Cl(-) cotransporters (NCCs)-and possibly two additional members-CCC interacting protein (CIP1) and polyamine transporters (CCC9)-as well. Altogether, CCCs can play essential physiological roles in transepithelial ion reabsorption and secretion, cell volume regulation, and inhibitory neurotransmission and so are present across all domains of life. To gain insight into the evolution of this family, we performed a comprehensive phylogenetic analysis using publically available genomic information. Our results clearly support CIP1 as being a true CCC based on shared evolutionary history. By contrast, the status of CCC9 in this regard remains equivocal. We also reveal the existence of a single ancestral CCC gene present in Archaea, from which numerous duplication events at the base of archaeans and eukaryotes lead to the divergence and subsequent neofunctionalization of the paralogous CCC subfamilies. A diversity of ensuing gene-loss events resulted in the complex distribution of CCCs present across the different taxa. Importantly, the occurrence of KCCs in "basal" metazoan taxa like sponges would allow an early formation of fast hyperpolarizing neurotransmission in metazoans. Gene duplications within the CCC subfamilies in vertebrates (in particular, KCCs, NKCCs, and NCCs) lend further evidence to the 2R hypothesis of two rounds of genome duplication at the base of the vertebrate lineage, especially in concert with our syntenic cluster analyses. This increased number of KCCs, NKCCs, and NCCs isoforms facilitates their further, important subfunctionalization in the vertebrate lineage.

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters.

The diuretic-sensitive cotransport of cations with chloride is mediated by the cation-chloride cotransporters, a large gene family encompassing a total of seven Na-Cl, Na-K-2Cl, and K-Cl cotransporters, in addition to two related transporters of unknown function. The cation-chloride cotransporters perform a wide variety of physiological roles and differ dramatically in patterns of tissue expression and cellular localization. The renal-specific Na-Cl cotransporter (NCC) and Na-K-2Cl cotransporter (NKCC2) are involved in Gitelman and Bartter syndrome, respectively, autosomal recessive forms of metabolic alkalosis. The associated phenotypes due to loss-of-function mutations in NCC and NKCC2 are consistent, in part, with their functional roles in the distal convoluted tubule and thick ascending limb, respectively. Other cation-chloride cotransporters are positional candidates for Mendelian human disorders, and the K-Cl cotransporter KCC3, in particular, may be involved in degenerative peripheral neuropathies linked to chromosome 15q14. The characterization of mice with both spontaneous and targeted mutations of several cation-chloride cotransporters has also yielded significant insight into the physiological and pathophysiological roles of several members of the gene family. These studies implicate the Na-K-2Cl cotransporter NKCC1 in hearing, salivation, pain perception, spermatogenesis, and the control of extracellular fluid volume. Targeted deletion of the neuronal-specific K-Cl cotransporter KCC2 generates mice with a profound seizure disorder and confirms the central role of this transporter in modulating neuronal excitability. Finally, the comparison of human and murine phenotypes associated with loss-of-function mutations in cation-chloride cotransporters indicates important differences in physiology of the two species and provides an important opportunity for detailed physiological and morphological analysis of the tissues involved.

The K+-Cl- cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na+-K+-2Cl- cotransporters and a thiazide-sensitive Na+-Cl- cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65-71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K+Cl- cotransporter: Cl--dependent uptake of 86Rb+ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K+-Cl- cotransport has implications for the physiology and pathophysiology of a number of tissues.

Chapter Six - Molecular Operation of the Cation Chloride Cotransporters: Ion Binding and Inhibitor Interaction

Ion concentration homeostasis is essential for normal neuronal functions and its changes can underlie different pathological conditions including seizures. However the mechanisms of these processes are poorly understood. Studying the dynamical and biophysical mechanisms of regulation of neuronal intra- and extra-cellular ion concentrations is important for the development of methods to treat neurological disorders. We constructed a conductance-based neuron model [1], which includes dynamic variables representing the intracellular Na+ and extracellular K+ concentrations. In this model, the Na+ and K+ concentrations are affected by sodium, potassium, and leak currents, Na+-K+ pump current, an uptake of potassium ions by glial current, and potassium diffusion. The leak current is represented by the sum of sodium, potassium and chloride leaks. The concentrations couple to the membrane voltage equations via the Nernst reversal potentials. The model also contains a voltage-gated calcium current and a simple model for the intracellular Ca2+ dynamics. The model produces slow and large-amplitude oscillations in ion concentrations similar to oscillations observable during seizures or seizure-like activity in vitro. We extended this model to include Cl- concentration dynamics. In the nervous system, the intracellular Cl- ion concentration (Cl-]i) determines the strength and polarity of GABAergic neurotransmission. This concentration is maintained by the activity of cation-chloride cotransporters (CCCs). We explored in a computational study the roles of two CCCs (the NA-K-2Cl cotransporter, NKCC1, and the K-Cl cotransporter, KCC2) in ion concentration homeostasis and in the generation of pathological oscillatory activity in neurons. Our computational studies show that reciprocal changes in the expression of NKCC1 (which elevates [Cl]i) and KCC2 (which decreases [Cl-]i) can change Cl- reversal potential (ECl) and significantly alter the effects of GABAA receptor (GABAAR) mediated inhibitory input. Under certain circumstances, this can evoke or prevent seizure-like activity, and we investigate dynamical and biophysical mechanisms of these phenomena. The model suggests that regulatory abilities of CCCs are increased with increasing GABAARs activation. Both simulated elevation of concentration of extracellular potassium ion ([K+]o,∞) and NA-K-2Cl cotransporter activity promote seizure-like activity. Our studies show that developmental regulation of expression of NKCC1 and KCC2, in conjunction with concentration dynamics, can alter Cl- electrochemical gradient and strength and polarity of GABAA neurotransmission. The computational studies corroborate that CCCs are potential targets for treatment of neurological diseases, which involve dysfunctions in intracellular ion concentration homeostasis.

The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days

Inflammation alters cation chloride cotransporter expression in sensory neurons

KCC2 membrane diffusion tunes neuronal chloride homeostasis

Editorial

Background: K-Cl cotransporters (KCCs) belong to the SLC12A cation/chloride cotransporters (CCCs) family that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. Regulation of KCCs activity by WNK3 kinase has been shown, where co-expresion with the kinase under hypotonic conditions; where KCCs are normally active, inhibits their transport. Whereas kinase dead WNK3 activates them under isotonic conditions where they are normally inhibited. Likewise, KCCs activation is abolished by inhibition of PP1A and PP2, demonstrating an essential regulatory role for PPs in this process. Substrate specificity for PPs is believed to be achieved by consensus binding motifs, present on their regulatory subunits stablishing a complex mechanism with several sensors, transducers, and effectors. KCCs regulation upon osmotic changes involves dephosphorylation of important regulatory sites in order to become fully activated, and on the other hand phosphorylation by WNK/SPAK/OSR1 kinases to become inhibited. In the present study we analyzed two putative PP1 binding sites present in the WNK3 kinase amino acid sequence to determine the role of WNK3 and protein phosphatase 1 (PP1) interaction upon KCCs phosphorylation/dephosphorylation regulation during cell volume and electrolyte homeostasis. Methods: KCCs activity was evaluated by Western blot analysis from HEK 293 cells transfected with KCC3, WNK3 WT, catalytically inactive WNK3DA, or PP1 binding sites mutants WNK3 PP1a, WNK3DA PP1A, WNK3 PP1B or WNK3DA PP1B. KCC3 dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated in KCCs regulatory sites Thr1039, Thr991 and Ser86. Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WNK3 kinase co-expression inhibited KCC3a Thr1039, Thr991 and Ser86 dephosphorylation under hypotonic conditions, eliminating their response to cell swelling. Nevertheless, when co-expressed with PP1 binding site mutant PP1a, KCC3 regulatory sites become dephosphorylated under isotonic and hypotonic conditions eliminating WNK3 inhibitory effect. WNK3 kinase autophosphorylation was evaluated in WT and PP1a mutant. Ser309 was phosphorylated in both osmotic conditions for WT WNK3, whereas PP1A mutant autophosphorylation was lost under isotonic and hypotonic environments. SPAK Thr373 regulation was unaffected in any of the conditions studied. Conclusions: Our results show a direct interaction between WNK3 and PP1 during KCCs regulation. We suggest WNK3 kinase inhibits PP1 activity, preventing KCC3 regulatory sites dephosphorylation. These data support the hypothesis of a phosphatase/kinase osmoregulatory complex controlling KCCs function during cell volume regulatory response. PAPPIT, Clave: IN222320 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.