A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength.

Benjamin König,Andrew Jr Plested,Tobias Stauber,Sophia Schwartz,Yuchen Hao

doi:10.7554/elife.45421

Abstract

Volume-regulated anion channels (VRACs) are central to cell volume regulation. Recently identified as hetero-hexamers formed by LRRC8 proteins, their activation mechanism remains elusive. Here, we measured Förster resonance energy transfer (FRET) between fluorescent proteins fused to the C-termini of LRRC8 subunits. Inter-subunit FRET from LRRC8 complexes tracked VRAC activation. With patch-clamp fluorometry, we confirmed that the cytoplasmic domains rearrange during VRAC opening. With these FRET reporters, we determined VRAC activation, non-invasively, in live cells and their subcompartments. Reduced intracellular ionic strength did not directly activate VRACs, and VRACs were not activated on endomembranes. Instead, pharmacological manipulation of diacylglycerol (DAG), and protein kinase D (PKD) activity, activated or inhibited plasma membrane-localized VRACs. Finally, we resolved previous contradictory reports concerning VRAC activation, using FRET to detect robust activation by PMA that was absent during whole-cell patch clamp. Overall, non-invasive VRAC measurement by FRET is an essential tool for unraveling its activation mechanism.

Highlights

Volume regulation is essential for virtually all cell types, to counteract osmotic swelling and shrinkage and during various processes like proliferation, migration and apoptosis (Hoffmann et al, 2009)
The same was observed when LRRC8A was combined with the Volume-regulated anion channels (VRACs) subunit LRRC8E (A-CFP/E-YFP or A-YFP/E-CFP), but not when combined with plasma membrane-localized CD4-YFP, used as control for Forster resonance energy transfer (FRET) by membrane crowding (Figure 1C)
The relationship of the observed FRET changes to VRAC gating are clear from numerous common features (Akita and Okada, 2014; Konig and Stauber, 2019; Pedersen et al, 2016; Strange et al, 2019): (i) the reversible decrease in FRET matches reversible VRAC activation upon treatment with hypo-osmotic solutions, and with the same kinetics as simultaneously-measured whole-cell VRAC currents, (ii) the intensity of the FRET decrease correlates with the applied extracellular hypotonicity; (iii) the modulation of VRAC currents, for example by cholesterol depletion, is mirrored by the observed FRET alteration; (iv) after activation with PMA, FRET rises in hypertonicity as VRAC is inactivated by cell shrinkage

Summary

Introduction

Volume regulation is essential for virtually all cell types, to counteract osmotic swelling and shrinkage and during various processes like proliferation, migration and apoptosis (Hoffmann et al, 2009). Volume-regulated anion channels (VRACs) are ubiquitously expressed and involved in regulatory volume decrease (RVD) of vertebrate cells (Chen et al, 2019; Jentsch, 2016; Stauber, 2015). These channels open slowly following osmotic cell swelling and mediate the extrusion of chloride and organic osmolytes, leading to the efflux of water and cell shrinkage. VRACs are composed of LRRC8 heteromers containing the essential subunit LRRC8A and at least one other LRRC8 protein, LRRC8B-E (Qiu et al, 2014; Voss et al, 2014). The C-terminal halves contain at least 16 leucine-rich repeats that form horseshoe-like structures reaching into the cytoplasm and their apparent flexibility

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