Structure and mechanism of the cation–chloride cotransporter NKCC1

Chew, Thomas; Orlando, Benjamin J.; Zhang, Jinru; Latorraca, Naomi R.; Wang, Amy; Hollingsworth, Scott A.; Chen, Donghua; Dror, Ron O.; Liao, Maofu; Feng, Liang

doi:10.1038/s41586-019-1438-2

Cited by 103 publications

(219 citation statements)

References 67 publications

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“…KCC4 is monomeric in the nanodisc structure, in contrast to the recent homodimeric structure of Danio rerio NKCC1 30 . Density for the N-terminal region and C-terminal domain (CTD), which together comprise approximately half the expressed protein mass, is not observed in the cryo-EM maps (Figure 2, S1, and S2).…”

Section: Resultsmentioning

confidence: 65%

“…Density for the N-terminal region and C-terminal domain (CTD), which together comprise approximately half the expressed protein mass, is not observed in the cryo-EM maps (Figure 2, S1, and S2). The N-terminal region is weakly conserved, variable in length among CCCs (Figure S1), and is likewise unresolved in the structure of NKCC1 30 . We presume it is highly flexible in KCC4.…”

Section: Resultsmentioning

confidence: 99%

“…We presume it is highly flexible in KCC4. The C-terminal domain, however, is well conserved, has documented roles in regulation, expression, and trafficking 1, 2,13,14,16 , and mediates homodimerization of NKCC1 and the Archaean CCC (MaCCC) 30, 31 .…”

Section: Resultsmentioning

confidence: 99%

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Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Reid

Kern

Brohawn

2019

Preprint

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AbstractCation-chloride-cotransporters (CCCs) catalyze transport of Cl− with K+ and/or Na+ across cellular membranes. CCCs play roles in volume regulation, neural development and function, audition, blood pressure regulation, and renal function. CCCs are targets of drugs including loop diuretics and their disruption is implicated in pathophysiologies including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the cryo-EM structure of a CCC, the Mus musculus K+-Cl− cotransporter KCC4, in lipid nanodiscs. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We further identify substrate K+ and Cl− binding sites and provide a structural explanation for differences in substrate specificity and transport ratio between CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

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Section: Resultsmentioning

confidence: 65%

Section: Resultsmentioning

confidence: 99%

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Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Reid

Kern

Brohawn

2019

Preprint

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“…A N-terminal loop is located between the scaffold and buddle domain ( Figure 1D). TM domain and CTD are connected through the Scissor Helix likewise in DrNKCC1 (39). The connection is stabilized by a salt bridge between the Scissor Helix and TM12 extension that are linked via a shorter hinge region (Supplemental Figure. Figure S3D).…”

Section: Kcc3 Architecture and Dimerizationmentioning

confidence: 99%

“…The recently reported DrNKCC1 and KCC1 structures provide the structural glimpse for the CCCs family (39,40). However, the limited sequence similarity between DrNKCC1 and KCCs as well as the lack of the cytosolic domain in the reported KCC1 structures hinder the elaboration of the KCCs working mechanism in detail.…”

Section: Introductionmentioning

confidence: 99%

Molecular basis for regulation of human potassium chloride cotransporters

Chi

Chen

et al. 2020

Preprint

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The Solute Carrier Family 12 (SLC12) encodes electroneutral cation-chloride cotransporters (CCCs) that are fundamental in cell volume regulation and chloride homeostasis. Dysfunction of CCCs engenders abnormality in renal function and neuro-system development. Here we presented structure of the full length human potassium-chloride co-transporter 2 (KCC2) and 3 (KCC3), the KCC3 mutants in phosphorylation mimic (P-mimic) and dephosphorylation mimic (DP-mimic) status, and KCC3 in complex with [(DihydroIndenyl)Oxy] Alkanoic acid (DIOA), a specific inhibitor of KCCs, at resolution of 2.7 Å -3.6 Å. A small N-terminal loop is bound at the intracellular vestibule of the transport path, arresting the transporter in an autoinhibition state. The C-terminal domain (CTD) of KCCs is structure-solved for the first time, revealing two conserved phosphorylation harboring segments (PHSs), which exhibit different conformation between P-mimic and DP-mimic mutants, explaining the inhibitory effect of phosphorylation. DIOA is located in between the two transmembrane domains, tightly bound to the loop between TM10 and TM11, locking the transporter in inward-facing conformation. Together, our study makes important steps in understanding the sophisticated regulation mechanisms of KCCs, with prospect for the specific drug development.

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An Induced Pluripotent Stem Cell‐Derived Neuromuscular Junction Platform for Study of the NGLY1‐Congenital Disorder of Deglycosylation

Sasserath

Robertson

Mendez

et al. 2022

Advanced Therapeutics

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There are many neurological rare diseases where animal models have proven inadequate or do not currently exist. NGLY1 deficiency, a congenital disorder of deglycosylation, is a rare disease that predominantly affects motor control, especially control of neuromuscular action. In this study, NGLY1-deficient, patient-derived induced pluripotent stem cells (iPSCs) are differentiated into motoneurons (MNs) to identify disease phenotypes analogous to clinical disease pathology with significant deficits apparent in the NGLY1-deficient lines compared to the control. A neuromuscular junction (NMJ) model is developed using patient and wild type MNs to study functional differences between healthy and diseased NMJs. Reduced axon length, increased and shortened axon branches, MN action potential (AP) bursting, and decreased AP firing rate and amplitude are observed in the NGLY1-deficient MNs in monoculture. When transitioned to the NMJ-coculture system, deficits in NMJ number, stability, failure rate, and synchronicity with indirect skeletal muscle stimulation are observed. This project establishes a phenotypic NGLY1 model for investigation of possible therapeutics and investigations into mechanistic deficits in the system.

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Structure and mechanism of the cation–chloride cotransporter NKCC1

Cited by 103 publications

References 67 publications

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Molecular basis for regulation of human potassium chloride cotransporters

An Induced Pluripotent Stem Cell‐Derived Neuromuscular Junction Platform for Study of the NGLY1‐Congenital Disorder of Deglycosylation

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