Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes

Gaitán‐Peñas, Héctor; Gradogna, Antonella; Laparra-Cuervo, Lara; Solsona, Carles; Fernández-Dueñas, Víctor; Barrallo-Gimeno, Alejandro; Ciruela, Francisco; Lakadamyali, Melike; Pusch, Michael; Estévez, Raúl

doi:10.1016/j.bpj.2016.08.030

Cited by 103 publications

(212 citation statements)

References 54 publications

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“…6 Xenopus oocytes were recently identified by us as an additional convenient expression system for detailed biophysical and pharmacological studies. 16 Oocytes possess a negligible background of volume-stimulated currents, and co-expression of LRRC8A with LRRC8C, LRRC8D, or LRRC8E induces hypotonicity activated currents with properties that are similar to those described in cellular systems. 6,16 In addition, we discovered that adding fluorescent proteins to the C-terminus of LRRC8 proteins induced constitutive anion currents that were further stimulated by hypotonicity, resulting in potentially very large currents that can be easily investigated.…”

Section: 17mentioning

confidence: 86%

“…4A, bottom). 16 Thus, overnight incubation of WT 8A/8E expressing oocytes in 1 mM cisplatin neither induced constitutive currents, nor increased hypotonicity activated currents (Fig. 4 A, B).…”

Section: Resultsmentioning

confidence: 99%

“…However, standard systems like HEK or CHO cells endogenously express VRAC, requiring LRRC8 gene-knockout cell lines for mechanistic investigations 6,15 We have recently found that Xenopus oocytes represent a convenient expression system for LRRC8 proteins. 16 Un-injected oocytes, or oocytes injected with single LRRC8 subunits had no detectable VRAC currents, whereas co-injection of LRRC8A and 8C, 8D or 8E (but not 8B) RNA yielded typical VRAC currents that slowly activated in hypotonic conditions. Furthermore, we discovered that the addition of fluorescent proteins to the C-terminus dramatically increased the activity: heteromeric LRRC8A-VFP/8C-mCherry, LRRC8A-VFP/8D-mCherry and LRRC8A-VFP/8E-mCherry exhibited considerable currents even in isotonic conditions, which were further strongly stimulated by hypotonicity.…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, we discovered that the addition of fluorescent proteins to the C-terminus dramatically increased the activity: heteromeric LRRC8A-VFP/8C-mCherry, LRRC8A-VFP/8D-mCherry and LRRC8A-VFP/8E-mCherry exhibited considerable currents even in isotonic conditions, which were further strongly stimulated by hypotonicity. 16 We performed a detailed electrophysiological characterization of these currents (ion selectivity, single channel analysis, radiotracer fluxes, blocker sensitivity). 16 Triggered by the proposed role of VRAC channels for the uptake of anticancer drugs, in the present addendum we exploited the oocyte expression system to test acute and chronic effects of the chemotherapeutic agent cisplatin.…”

Section: Introductionmentioning

confidence: 99%

“…16 We performed a detailed electrophysiological characterization of these currents (ion selectivity, single channel analysis, radiotracer fluxes, blocker sensitivity). 16 Triggered by the proposed role of VRAC channels for the uptake of anticancer drugs, in the present addendum we exploited the oocyte expression system to test acute and chronic effects of the chemotherapeutic agent cisplatin. We found that overnight incubation with cisplatin dramatically increases currents in oocytes that express heteromeric LRRC8A-VFP/ 8D-mCherry or LRRC8A-VFP/8E-mCherry.…”

Section: Introductionmentioning

confidence: 99%

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Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

et al. 2017

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LRRC8 proteins have been shown to underlie the ubiquitous volume regulated anion channel (VRAC). VRAC channels are composed of the LRRC8A subunit and at least one among the LRRC8B-E subunits. In addition to their role in volume regulation, LRRC8 proteins have been implicated in the uptake of chemotherapeutic agents. We had found that LRRC8 channels can be conveniently expressed in Xenopus oocytes, a system without endogenous VRAC activity. The fusion with fluorescent proteins yielded constitutive activity for A/C, A/D and A/E heteromers. Here we tested the effect of the anticancer drug cisplatin on LRRC8A-VFP/8E-mCherry and LRRC8A-VFP/8D-mCherry co-expressing oocytes. Incubation with cisplatin dramatically activated currents for both subunit combinations, confirming that VRAC channels provide an uptake pathway for cisplatin and that intracellular cisplatin accumulation strongly activates the channels. Thus, specific activators of LRRC8 proteins might be useful tools to counteract chemotherapeutic drug resistance.

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Section: 17mentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

et al. 2017

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The Pannexin1 membrane channel: distinct conformations and functions

Dahl

2018

FEBS Letters

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The Pannexin1 (Panx1) membrane channel responds to different stimuli with distinct channel conformations. Most stimuli induce a large cation- and ATP-permeable conformation, hence Panx1 is involved in many physiological processes entailing purinergic signaling. For example, oxygen delivery in the peripheral circulatory system is regulated by ATP released from red blood cells and endothelial cells through Panx1 channels. The same membrane channel, however, when stimulated by positive membrane potential or by cleavage with caspase 3, is highly selective for the passage of chloride ions, excluding cations and ATP. Although biophysical data do not allow a distinction between the chloride-selective channels induced by voltage or by caspase cleavage, there must be other subtle differences in the structure, because overexpression of wtPanx1 is well tolerated by cells, while expression of the truncation mutant Panx1Δ378 results in slow cell death. Thus, in addition to the well-characterized two open conformations, there might be a third, more subtle conformational change involved in cell death.

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“Find Me” and “Eat Me” signals: tools to drive phagocytic processes for modulating antitumor immunity

Xiao,

Zhang,

Guo

et al. 2024

Cancer Communications

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Phagocytosis, a vital defense mechanism, involves the recognition and elimination of foreign substances by cells. Phagocytes, such as neutrophils and macrophages, rapidly respond to invaders; macrophages are especially important in later stages of the immune response. They detect “find me” signals to locate apoptotic cells and migrate toward them. Apoptotic cells then send “eat me” signals that are recognized by phagocytes via specific receptors. “Find me” and “eat me” signals can be strategically harnessed to modulate antitumor immunity in support of cancer therapy. These signals, such as calreticulin and phosphatidylserine, mediate potent pro‐phagocytic effects, thereby promoting the engulfment of dying cells or their remnants by macrophages, neutrophils, and dendritic cells and inducing tumor cell death. This review summarizes the phagocytic “find me” and “eat me” signals, including their concepts, signaling mechanisms, involved ligands, and functions. Furthermore, we delineate the relationships between “find me” and “eat me” signaling molecules and tumors, especially the roles of these molecules in tumor initiation, progression, diagnosis, and patient prognosis. The interplay of these signals with tumor biology is elucidated, and specific approaches to modulate “find me” and “eat me” signals and enhance antitumor immunity are explored. Additionally, novel therapeutic strategies that combine “find me” and “eat me” signals to better bridge innate and adaptive immunity in the treatment of cancer patients are discussed.

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Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes

Cited by 103 publications

References 54 publications

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

Cisplatin activates volume sensitive LRRC8 channel mediated currents in Xenopus oocytes

The Pannexin1 membrane channel: distinct conformations and functions

“Find Me” and “Eat Me” signals: tools to drive phagocytic processes for modulating antitumor immunity

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