Structure of the human volume regulated anion channel

Kefauver, Jennifer M.; Saotome, Kei; Dubin, Adrienne E.; Pallesen, Jesper; Cottrell, Christopher A.; Hong, Gunhee; Cahalan, Stuart M.; Qiu, Zhaozhu; Crowley, Catherine; Whitwam, Tess; Lee, Wen-Hsin; Ward, Andrew B.; Patapoutian, Ardem

doi:10.1101/323584

Cited by 18 publications

(47 citation statements)

References 41 publications

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“…VRAC operates as a heteromeric complex of several proteins from the LRRC8 family. Since the molecular structure of VRAC was identified, a series of antibodies against different regions of the basic subunits of the family LRRC8 have been developed and effectively used [4,5,9,14,17,18,[23][24][25][26]. Before now, VRAC subunit proteins were detected mostly by immunoblotting of the solubilized whole cells [27][28][29][30][31][32], less often of cell membranes lysates [14,33,34] or by imaging cells that have been permeabilized for staining [35,36].…”

Section: Discussionmentioning

confidence: 99%

“…A growing body of evidence indicate that VRAC and their obligatory subunit, LRRC8A have critical roles in many cell functions including cell motility, proliferation, apoptosis, drug and metabolite transport, angiogenesis, and spermatid development, as well as in cell pathophysiological cell functions such as cancer drug resistance, ischemic brain edema, and glaucoma [7][8][9][10][11][12][13][14][15][16][17]. While the molecular structure of VRAC is well documented [18], understanding how VRAC expression at the membrane is regulated has been poorly explored due to lack of appropriate methodology. Electrophysiological methods are suitable for investigating biophysical properties of channels in a single cell and during short-time events but are unsuitable for studying cell populations and the long-term alteration of cells.…”

Section: Introductionmentioning

confidence: 99%

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Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

et al. 2020

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Assessing the expression of channels on the cell membrane is a necessary step in studying the functioning of ion channels in living cells. We explore, first, if endogenous VRAC can be assayed using flow cytometry and a commercially available antibody against an extracellular loop of the LRRC8A, also known as SWELL1, subunit of the VRAC channel. The second goal is to determine if an increase in the number of VRAC channels at the cell membrane is responsible for an increase in chloride permeability of the membrane in two well-known cases: during staurosporine (STS)induced apoptosis and after water balance disturbance caused by hypotonic medium. Human suspension lymphoid cells U937 were used as they are suitable for flow fluorometry and because we have recently studied their membrane chloride permeability during apoptosis. We found that surface expression of endogenous LRRC8A subunits can be quantified in living U937 cells using flow fluorometry with the Alomone Lab antibody. Further, we revealed that treatment of cells for 1 hour using STS or a hypotonic solution did not change the number of LRRC8A subunits to the extent that would correspond to changes in the membrane chloride permeability determined by ion content analysis. This indicates that prolonged increase in chloride permeability of the cell membrane during apoptotic cell shrinkage or cell volume regulation under hypotonicity in U937 cells occurs without altering cell surface expression of VRAC.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

et al. 2020

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“…The LRRC8 protein family embraces the five members LRRC8A/B/C/D/E, which are all membrane-spanning proteins composed of four trans-membrane domains and a long C-terminal leucine-rich repeat domain (LRRD) [40,41]. As sequence analyses indicated that the LRRC8 family are related to pannexins’ channel complexes, which release larger signaling molecules as ATP from the cytoplasm to the extracellular environment [42,43], it has been suggested that LRRC8 proteins may form multimeric channel complexes with varying LRRC8 protein stoichiometry [41,42].…”

Section: Cellular Cisplatin Accumulationmentioning

confidence: 99%

“…As sequence analyses indicated that the LRRC8 family are related to pannexins’ channel complexes, which release larger signaling molecules as ATP from the cytoplasm to the extracellular environment [42,43], it has been suggested that LRRC8 proteins may form multimeric channel complexes with varying LRRC8 protein stoichiometry [41,42]. Protein sequence analysis has revealed that LRRC8A contains putative cisplatin-interacting methionines in the cellular and transmembrane segments as well as CxxC/YxxY motifs that can be oxidized/phosphorylated, but their significance for the facilitation of cisplatin uptake has yet to be revealed.…”

Section: Cellular Cisplatin Accumulationmentioning

confidence: 99%

“…In summary, decreased cellular cisplatin accumulation in resistant cells can reflect reduced uptake through CTR1 and LRRC8A/8D-dependent VRACs, as well as increased cisplatin release through MRP, and the ATPases ATP7A and ATP7B. As reversible downregulation of VRAC activity may in some cell lines involve alteration of the membrane and/or total expression of LRRC8A/LRRC8D, our current knowledge to the cellular signaling involved in the regulation of VRAC activity [31,32,38,39] and the recent revelation of the LRRC8A structure using high-resolution cryo-electron microscopy [41] raises the possibility of controlling cisplatin uptake through the manipulation of the selectivity/open-probability of the VRAC complex.…”

Section: Cellular Cisplatin Accumulationmentioning

confidence: 99%

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Facilitating the Cellular Accumulation of Pt-Based Chemotherapeutic Drugs

Lambert

Sørensen

2018

IJMS

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Cisplatin, carboplatin, and oxaliplatin are Pt-based drugs used in the chemotherapeutic eradication of cancer cells. Although most cancer patient cells initially respond well to the treatment, the clinical effectiveness declines over time as the cancer cells develop resistance to the drugs. The Pt-based drugs are accumulated via membrane-bound transporters, translocated to the nucleus, where they trigger various intracellular cell death programs through DNA interaction. Here we illustrate how resistance to Pt-based drugs, acquired through limitation in the activity/subcellular localization of canonical drug transporters, might be circumvented by the facilitated uptake of Pt-based drug complexes via nanocarriers/endocytosis or lipophilic drugs by diffusion.

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Molecular Biology and Physiology of Volume-Regulated Anion Channel (VRAC)

Osei-Owusu

Yang

Vitery

et al. 2018

Current Topics in Membranes

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The Volume-Regulated Anion Channel (VRAC) is activated by cell swelling and plays a key role in cell volume regulation. VRAC is ubiquitously expressed in vertebrate cells and also implicated in many other physiological and cellular processes including fluid secretion, glutamate release, membrane potential regulation, cell proliferation, migration, and apoptosis. Although its biophysical properties have been well characterized, the molecular identity of VRAC remained a mystery for almost three decades. The field was transformed by recent discoveries showing that the leucine-rich repeat-containing protein 8A (LRRC8A, also named SWELL1) and its four other homologs form heteromeric VRAC channels. The composition of LRRC8 subunits determines channel properties and substrate selectivity of a large variety of different VRACs. Incorporating purified SWELL1-containing protein complexes into lipid bilayers is sufficient to reconstitute channel activities, a finding that supports the decrease in intracellular ionic strength as the mechanism of VRAC activation during cell swelling. Characterization of Swell1 knockout mice uncovers the important role of VRAC in T cell development, pancreatic β-cell glucose-stimulated insulin secretion, and adipocyte metabolic function. The ability to permeate organic osmolytes and metabolites is a major feature of VRAC. The list of VRAC substrates is expected to grow, now also including some cancer drugs and antibiotics even under non-cell swelling conditions. Therefore, a critical role of VRAC in drug resistance and cell-cell communication is emerging. This review summarizes the exciting recent progress on the structure-function relationship and physiology of VRAC and discusses key future questions to be solved.

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Structure of the human volume regulated anion channel

Cited by 18 publications

References 41 publications

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

Flow fluorometry quantification of anion channel VRAC subunit LRRC8A at the membrane of living U937 cells

Facilitating the Cellular Accumulation of Pt-Based Chemotherapeutic Drugs

Molecular Biology and Physiology of Volume-Regulated Anion Channel (VRAC)

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