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The thiazide sensitive Na+:Cl− cotransporter (NCC) is the principal via for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema. NCC was the first member of the electroneutral cation-coupled chloride cotransporter family to be identified at a molecular level. It was cloned from the urinary bladder of the Pseudopleuronectes americanus (winter flounder) 30 years ago. The structural topology, kinetic and pharmacology properties of NCC have been extensively studied, determining that the transmembrane domain (TM) coordinates ion and thiazide binding. Functional and mutational studies have discovered residues involved in the phosphorylation and glycosylation of NCC, particularly on the N-terminal domain, as well as the extracellular loop connected to TM7-8 (EL7-8). In the last decade, single-particle cryogenic electron microscopy (cryo-EM) has permitted the visualization of structures at high atomic resolution for six members of the SLC12 family (NCC, NKCC1, KCC1-KCC4). Cryo-EM insights of NCC confirm an inverted conformation of the TM1-5 and TM6-10 regions, a characteristic also found in the amino acid-polyamine-organocation (APC) superfamily, in which TM1 and TM6 clearly coordinate ion binding. The high-resolution structure also displays two glycosylation sites (N-406 and N-426) in EL7-8 that are essential for NCC expression and function. In this review, we briefly describe the studies related to the structure-function relationship of NCC, beginning with the first biochemical/functional studies up to the recent cryo-EM structure obtained, to acquire an overall view enriched with the structural and functional aspects of the cotransporter.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC’s activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.

The role of Cl– as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl– in signaling is the modulation of the With-No-Lysine (K) (WNK) – STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) – Cation-Coupled Cl–Cotransporters (CCCs) cascade. Binding of a Cl– anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl– release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na+-driven CCCs (mediating ions influx) promote their activation, whereas that of the K+-driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl– influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl– sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.