Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling

Moseng, Mitchell A.; Su, Chih‐Chia; Rios, Kerri; Cui, Meng; Lyu, Meinan; Glaza, Przemysław; Klenotic, Philip A.; Delpire, Eric; Yu, Edward

doi:10.1126/sciadv.abq0952

Cited by 14 publications

(34 citation statements)

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“…Here, we found that these hydrophobic interactions involved Ala 414, Val 417, Val 418 and Leu 421 from TM 4, while Phe 659, Leu 663 and Ile 666 from TM 9 (Figure ). These interactions, statically present also in the cryo-EM structures, − were stably maintained during our equilibrium MD simulations, in both the IO and OO states. In addition, we observed the crucial involvement of TM 10, which was key to the NKCC1’s rocking-bundle mechanism in our simulations.…”

Section: Resultsmentioning

confidence: 64%

“…Interestingly, the hydration level of the outer vestibule in the OO state was significantly affected by the Arg 307–Glu 389 salt bridge (Figure S8). It is also worth noting that the drug bumetanide is bound to the outer vestibule in all the available cryo-EM structures of NKCC1 in the OO state. , Ligand binding at the outer vestibule hampers the formation of the Arg 307-Glu 389 salt bridge, which is, therefore, proven to be crucial for the rocking-bundle mechanism in NKCC1.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, 15 NKCC1 structures have recently been resolved alone or together with unselective inhibitors (e.g., the FDA-approved diuretic bumetanide). All these recent cryo-EM structures of NKCC1 have clarified several functional features of its multidomain system, − revealing a structural similarity to the LeuT-fold transporters. , In particular, NKCC1 is a homodimer, where each monomer is constructed by three main components: a conserved transmembrane (TM) domain composed of 12 helices organized in two inverted repeats of five-helix bundles, which contain all four ion binding sites (Figure A). Two additional intracellular domains are the disordered amino-terminal and the large carboxy-terminal domains, which are related to NKCC1 activation (Figure B) …”

Section: Introductionmentioning

confidence: 99%

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Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism

Ruiz Munevar,

Rizzi,

Portioli

et al. 2023

J. Am. Chem. Soc.

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The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na + -dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

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Section: Resultsmentioning

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism

Ruiz Munevar,

Rizzi,

Portioli

et al. 2023

J. Am. Chem. Soc.

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“…A qualitative analysis of this latter BUM binding mode to the outward-facing NKCC1 ( Figure S13, Table S8 ) shows that BUM in this binding pose exhibits a binding free energy comparable to that obtained in this study. In addition, while we were revising the manuscript, a new structure of the human NKCC1 was released showing yet another possible binding site of the loop diuretics located in the intracellularly-exposed C-terminal domain [ 43 ]. This new finding supports the proposition that BUM can cross the cell membrane and bind to NKCC1 from the cytosolic side and suggests that the loop diuretic binding modes/mechanism are perhaps more intricate than originally envisaged.…”

Section: Discussionmentioning

confidence: 99%

Role of Monovalent Ions in the NKCC1 Inhibition Mechanism Revealed through Molecular Simulations

Janoš

Magistrato

2022

IJMS

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The secondary active Na-K-Cl cotransporter 1 (NKCC1) promotes electroneutral uptake of two chloride ions, one sodium ion and one potassium ion. NKCC1 regulates Cl− homeostasis, thus being implicated in transepithelial water transport and in neuronal excitability. Aberrant NKCC1 transport is linked to a variety of human diseases. The loop diuretic drugs bumetanide, furosemide, azosemide and ethacrynic acid target NKCC1, but are characterized by poor selectivity leading to severe side effects. Despite its therapeutic importance, the molecular details of the NKCC1 inhibition mechanism remain unclear. Using all-atom simulations, we predict a putative binding mode of these drugs to the zebrafish (z) and human (h) NKCC1 orthologs. Although differing in their specific interactions with NKCC1 and/or monovalent ions, all drugs can fit within the same cavity and engage in hydrophobic interactions with M304/M382 in z/hNKCC1, a proposed ion gating residue demonstrated to be key for bumetanide binding. Consistent with experimental evidence, all drugs take advantage of the K+/Na+ ions, which plastically respond to their binding. This study not only provides atomic-level insights useful for drug discovery campaigns of more selective/potent NKCC1 inhibitors aimed to tackle diseases related to deregulated Cl− homeostasis, but it also supplies a paradigmatic example of the key importance of dynamical effects when drug binding is mediated by monovalent ions.

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“…Like the Na–K–2Cl cotransporter [5], the K–Cl cotransporter functional unit is a dimer [6]. The recent cryo-electron microscopy (cryo-EM) structures of NKCC1 [7 ▪▪ ,8 ▪ ], KCC1–KCC4 [9 ▪▪ ,10,11] confirms the dimeric nature of the transporters. As cells express multiple K–Cl cotransporters [12–14], it is believed that they likely express heterodimers.…”

Section: Introductionmentioning

confidence: 87%

The K–Cl cotransporter-3 in the mammalian kidney

Ferdaus,

Delpire

2023

Current Opinion in Nephrology &Amp; Hypertension

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Purpose of review We recently localized a new K–Cl cotransporters-3 (KCC3) transporter to the apical membrane of type-B intercalated cells. This gives us an opportunity to revisit the roles of the KCC3 in kidney and integrate the new findings to our current knowledge of the biology of the bicarbonate secreting cells. Recent findings Here, we review the basic properties of the K–Cl cotransporter with a particular attention to the responsiveness of the transporter to cell swelling. We summarize what is already known about KCC3b and discuss new information gained from our localizing of KCC3a in type-B intercalated cells. We integrate the physiology of KCC3a with the main function of the type-B cell, that is, bicarbonate secretion through the well characterized apical Cl−/HCO3 − exchanger and the basolateral Na-HCO3 cotransporter. Summary Both KCC3b and KCC3a seem to be needed for maintaining cell volume during enhanced inward cotransport of Na-glucose in proximal tubule and Na-HCO3 in intercalated cells. In addition, apical KCC3a might couple to pendrin function to recycle Cl−, particularly in conditions of low salt diet and therefore low Cl− delivery to the distal tubule. This function is critical in alkalemia, and KCC3a function in the pendrin-expressing cells may contribute to the K+ loss which is observed in alkalemia.

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Inhibition mechanism of NKCC1 involves the carboxyl terminus and long-range conformational coupling

Cited by 14 publications

References 50 publications

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism

Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism

Role of Monovalent Ions in the NKCC1 Inhibition Mechanism Revealed through Molecular Simulations

The K–Cl cotransporter-3 in the mammalian kidney

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