The expanding toolbox to study the LRRC8-formed volume-regulated anion channel VRAC.

The volume-regulated anion channel (VRAC) is activated upon cell swelling and facilitates the passive movement of anions across the plasma membrane in cells. VRAC function underlies many critical homeostatic processes in vertebrate cells. Among them are the regulation of cell volume and membrane potential, glutamate release and apoptosis. VRAC is also permeable for organic osmolytes and metabolites including some anti-cancer drugs and antibiotics. Therefore, a fundamental understanding of VRAC's structure-function relationships, its physiological roles, its utility for therapy of diseases, and the development of compounds modulating its activity are important research frontiers. Here, we describe approaches that have been applied to study VRAC since it was first described more than 30 years ago, providing an overview of the recent methodological progress. The diverse applications reflecting a compromise between the physiological situation, biochemical definition, and biophysical resolution range from the study of VRAC activity using a classic electrophysiology approach, to the measurement of osmolytes transport by various means and the investigation of its activation using a novel biophysical approach based on fluorescence resonance energy transfer.

Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting.

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents.

More than just a pressure relief valve: physiological roles of volume-regulated LRRC8 anion channels.

The volume-regulated anion channel (VRAC) is a key player in the volume regulation of vertebrate cells. This ubiquitously expressed channel opens upon osmotic cell swelling and potentially other cues and releases chloride and organic osmolytes, which contributes to regulatory volume decrease (RVD). A plethora of studies have proposed a wide range of physiological roles for VRAC beyond volume regulation including cell proliferation, differentiation and migration, apoptosis, intercellular communication by direct release of signaling molecules and by supporting the exocytosis of insulin. VRAC was additionally implicated in pathological states such as cancer therapy resistance and excitotoxicity under ischemic conditions. Following extensive investigations, 5 years ago leucine-rich repeat-containing family 8 (LRRC8) heteromers containing LRRC8A were identified as the pore-forming components of VRAC. Since then, molecular biological approaches have allowed further insight into the biophysical properties and structure of VRAC. Heterologous expression, siRNA-mediated downregulation and genome editing in cells, as well as the use of animal models have enabled the assessment of the proposed physiological roles, together with the identification of new functions including spermatogenesis and the uptake of antibiotics and platinum-based cancer drugs. This review discusses the recent molecular biological insights into the physiology of VRAC in relation to its previously proposed roles.

Determination of oxygen derived free radicals producer (xanthine oxidase) and scavenger (paraoxonase1) enzymes and lipid parameters in different cancer patients.

BACKGROUND: Cancer is a major cause of death throughout the world. Xanthine oxidase (XO) is considered an oxygen derived free radicals producer and paraoxonasel (PON1) is known as a free radicals scavenger. This study was undertaken to determine the activity of PON1 and XO and their concentration along with the lipid profile, lipid peroxide, and thiol level in cancer patients and healthy persons. METHODS: Antigen competitive ELISA was used to determine the level of paraoxonasel and xanthine oxidase in plasma. Paraoxonase and arylesterase activity of the PON1 enzyme and the activity of the XO enzyme were also determined. PON1 and XO activity, cholesterol, LDL-C, HDL-C, lipid peroxides, and thiol level in patients with breast cancer (n = 50), prostate cancer (n = 20), lung cancer (n = 12,), cervix cancer (n = 15), Non-Hodgkin lymphoma (n = 10), and acute lymphoblastic lymphoma (n = 8) and total age matched healthy persons (n = 115) were studied. RESULTS: Low plasma paraoxonase/arylesterase activities (p < 0.05) and the concentration of the PON1 enzyme were observed in all cancer patients. An elevated level of XO activity was observed in cancer patients. Among different cancers, comparatively high level of XO activity was noted in acute lymphoblastic lymphoma patients (0.18 +/- 0.03, p = 0.001) except cervix cancer patients (0.053 +/- 0.03, p = 0.0029) where a low level was observed. Low HDL-C (p < 0.01), cholesterol (p < 0.01), triglycerides (p < 0.01), thiol levels (p < 0.01) and high lipid peroxide levels (p > 0.05) were observed in cancer patients. CONCLUSIONS: Lower PON1 and high XO enzyme activities cause an imbalance of the free radical system which enhances the lipid peroxidation and other pathological conditions. Lower HDL-C level is also indicative of the low level of PON1 enzyme activity.