Gaitán-Peñas H , Gradogna A , Laparra-Cuervo L , Solsona C , Fernández-Dueñas V , Barrallo-Gimeno A , Ciruela F , Lakadamyali M , Pusch M , Estévez R .

GGP12008 Telethon, TI_GGP12008 Telethon

	Figure 1. Functional expression of LRRC8-mediated VRAC currents in Xenopus oocytes. (A) Voltage-clamp traces of an uninjected oocyte evoked by the IV-pulse protocol in “Iso” (A, top left) and after 5 min perfusion of “Hypo” solution (A, top right). Mean values of currents at 60 mV from uninjected oocytes in “Iso,” and after 5 min and 10 min in “Hypo” solution (A, bottom) (n = 7). (B–G, top) Currents of single oocytes injected with 8A (B) and 8E (C), and coinjected with 8A/8E (D), 8A-VFP/8E-mCherry (E), 8A/8E-mCherry (F), and 8A-VFP/8E (G) in response to the IV-pulse protocol in “Iso” solution and after 5 min perfusion with “Hypo” solution. (B–G, bottom) Current at 60 mV of 8A (B), 8E (C), 8A/8E (D), 8A-VFP/8E-mCherry (E), 8A/8E-mCherry (F), and 8A-VFP/8E (G) in “Iso” and after 5 min and 10 min in “Hypo” solution (n ≥ 4). The dashed line in (A)–(D) indicates an arbitrary threshold of current expression corresponding to 0.5 μA. (H and I) Mean values of currents at 60 mV from oocytes injected with 8A-VFP (H) and 8E-mCherry (I) in “Iso” and after 5 min and 10 min in “Hypo” solution (n ≥ 4). Error bars indicate the standard deviation.
	Figure 2. Inactivation and selectivity properties of VRAC currents mediated by different combinations of LRRC8 proteins. (A–D) Comparison of the current magnitude induced by co-expressing 8A-VFP with 8B-mCherry (A), 8C-mCherry (B), 8D-mCherry (C), and 8E-mCherry (D). Top: current traces from single oocytes evoked by the IV-pulse protocol in “Iso” solution (left) and after 5 min perfusion with “Hypo” solution (right). Middle: voltage clamp traces shown at higher magnification illustrate the typical relaxation kinetics. Horizontal bars: 1 sec; vertical bars: indicated in figure. Bottom: mean currents recorded in “Iso” solution, and after 5 min and 10 min in “Hypo” solution (n ≥ 4). (E) Permeation properties of hLRRC8A-VFP co-expressed with 8E-mCherry (left), 8C-mCherry (middle) and 8D-mCherry (right). Permeability ratios are estimated from the shifts in Erev caused by the substitution of Cl− with glu, glutamate; gluc, gluconate; asp, aspartate; lact, lactate; tau, taurine; gly, glycine; HCO3, bicarbonate; F, fluoride; Br, bromide; NO3, nitrate; I, iodide; SCN, thiocyanate, or Na+ with TEA (n ≥ 4 for 8A-VFP/8E-mCherry; n ≥ 4; except for bicarbonate n = 3 for 8A-VFP/8C-mCherry; n ≥ 3 for 8A-VFP/8D-mCherry). The dashed line indicates the permeability for chloride. Primary data are shown in Fig. S7. Error bars indicate SD.
	Figure 3. Single-channel studies of the 8A-VFP/8E-mCherry heteromer. (A) Representative traces from a cell-attached patch at 80, 40, and −80 mV. (B) Single-channel current-voltage relationship. Mean single-channel currents are plotted versus the corresponding potentials ranging between −80 and 80 mV. Note the outward rectification (n ≥ 4) For −80, −40, and −20 mV, n = 2). Error bars indicate the standard deviation. (C) Amplitude histogram of the recording at 80 mV in control conditions. The dashed line represents the fit with a sum of three Gaussians. The dominant current level has an amplitude of 2.5 pA. To see this figure in color, go online.
	Figure 4. Uptake of osmolytes through LRRC8 proteins in Xenopus oocytes. (A and B) Comparison of the mean values of taurine (A) and glutamate (B) uptake between uninjected (n ≥ 6) oocytes, oocytes injected with nontagged 8A/8E (n ≥ 5) and oocytes injected with fluorescent-tagged 8A-VFP/8E-mCherry (n ≥ 6) in “Iso” solution (left) and “Hypo” solution (right). (C–E) Comparison of the mean values of taurine (C), glutamate (D), and glycine (E) uptake between uninjected oocytes and oocytes injected with 8A-VFP co-expressed with 8B-mCherry, 8C-mCherry, 8D-mCherry, and 8E-mCherry (n ≥ 3 for all co-expressions) in Iso “I” and Hypo “H” solution. (F) Time course of normalized oocyte volume in ND48 solution and apparent water permeability mean values (inset) of uninjected oocytes (n = 6), AQP1 + 8A-VFP (n = 8), and AQP1 + 8A-VFP/8E-mCherry (n = 10) injected oocytes. Data indicate the mean ± SE. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 5. ATP release through LRRC8 proteins. (A) ATP-release representative traces of uninjected oocytes (bottom), oocytes injected with 8A/8E (middle), and oocytes injected with 8A-VFP/8E-mCherry (top). The black arrowhead indicates the beginning of the hypotonic exposure (15 mOsm). (B) Amount of ATP released (pmol/oocyte/min) from uninjected oocytes (n = 4), oocytes injected with 8A/8E (n = 5) and oocytes injected with 8A-VFP/8E-mCherry (n = 5). (C) Amount of ATP released from oocytes injected with 8A-VFP/8E-mCherry incubated in solutions with different osmolarities, 15 (n = 6), 74 (n = 4), and 196 (n = 4) mOsm, or from uninjected oocytes at 15 mOsm (n = 4). (D) Amount of ATP released in hypotonic conditions from oocytes injected with 8A-VFP/8E-mCherry (n = 6), 8A-VFP/8E-mCherry in the presence of 100 μM CBX (n = 5), 8A-VFP/8C-mCherry (n = 4), 8A-VFP/8B-mCherry (n = 4), 8A-VFP/8D-mCherry (n = 4), and uninjected oocytes (n = 4). Data indicate the mean ± SE. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 6. Carbenoxolone inhibition of 8A-VFP/8E-mCherry. (A) Currents recorded in voltage clamp at 60 mV are plotted as function of the time. Colors correspond to the different CBX concentrations (10, 20, 50, 100, 500 μM) applied during the experiment. In order to estimate the steady-state effect, single-exponential functions were fitted to the time course and extrapolated to infinite time (solid colored lines). (B) Concentration dependence of CBX modulation of hLRRC8A-VFP/8E-mCherry. Currents acquired at 60 mV are normalized to values measured in “Iso” solution and plotted versus CBX concentrations. The red line represents the best fit of hLRRC8A-VFP/8E-mCherry currents (fit parameters: Hill coefficient = 1.8, KA = 11.9 μM, p∞ = 0.038), while the black dashed line (fit parameters: Hill coefficient = 1.0, KA = 10.6 μM, p∞ = 0) poorly fits the data. These theoretical predictions are obtained using Eq. 3 as described in Methods. (n ≥ 8, except for 50 μM, where n = 4). (C) Representative trace from an outside-out patch at 80 mV in “Iso” solution (black), 100 μM CBX (red), and wash in “Iso” solution (black). Error bars indicate SD. To see this figure in color, go online.
	Figure 7. Subunit counting of LRRC8 heteromers by single-step photobleaching. (A) TIRF image showing LRRC8A-VFP and LRRC8E-mCherry oligomers on the Xenopus oocyte membrane and a merge of the two fluorescent channels. Arrowheads point to overlapping LRRC8A and LRRC8E spots. Scale bar: 5 μm. (B) Intensity-time traces showing individual photobleaching steps from one of the LRRC8A-VFP oligomers in complex with LRRC8E-mCherry (upper) or the LRRC8E-VFP oligomers in complex with LRRC8A-mCherry (lower) identified on the oocyte membrane in TIRF microscopy. Dashed lines and arrows indicate observed single steps. (C) Distribution of the percentage of counted steps (n = 142) from LRRC8A-VFP oligomers in complex with LRRC8E-mCherry oligomers at equimolar ratio. (D) Distribution of the percentage of counted steps (n = 158) from LRRC8E-VFP oligomers in complex with LRRC8A-mCherry oligomers at equimolar ratio. Blue bars in C and D indicate the data, orange bars show the prediction of a binomial distribution (Eq. 4) with P = 0.8 and gray bars indicate the best fits of Poisson distribution (Eq. 5). To see this figure in color, go online.
	Figure 8. Osmolarity dependence of 8A-VFP/8E-mCherry. Left: currents at 60 mV are plotted vs. time. Colors correspond to the different extracellular osmolarities applied (200, 310, 120 mOsm); additionally, CBX was applied to test the endogenous current contribution. Right: quantification of the osmolarity dependence of 8A-VFP/8E-mCherry. Currents at 60 mV recorded at osmolarity values between 40 and 310 mOsm are normalized to the currents measured in “Iso” solution (200 mOsm). Error bars indicate SD (n ≤ 5). To see this figure in color, go online.

Abascal, LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication. 2012, Pubmed

Abascal, LRRC8 proteins share a common ancestor with pannexins, and may form hexameric channels involved in cell-cell communication. 2012, Pubmed
Ambrosi, Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. 2010, Pubmed , Xenbase
Arellano, Functional role of follicular cells in the generation of osmolarity-dependent Cl- currents in Xenopus follicles. 1995, Pubmed , Xenbase
Benfenati, Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia. 2009, Pubmed
Birkenhäger, A novel homozygous mutation in the EC1/EC2 interaction domain of the gap junction complex connexon 26 leads to profound hearing impairment. 2014, Pubmed
Blumenthal, The minK potassium channel exists in functional and nonfunctional forms when expressed in the plasma membrane of Xenopus oocytes. 1994, Pubmed , Xenbase
Boassa, Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane. 2007, Pubmed
Bröer, Xenopus laevis Oocytes. 2010, Pubmed , Xenbase
Bumb, Quantitative characterization of fluorophores in multi-component nanoprobes by single-molecule fluorescence. 2011, Pubmed
Chillarón, Pathophysiology and treatment of cystinuria. 2010, Pubmed
Decher, DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration. 2001, Pubmed , Xenbase
de Pina-Benabou, Blockade of gap junctions in vivo provides neuroprotection after perinatal global ischemia. 2005, Pubmed
Dourado, Pannexin-1 is blocked by its C-terminus through a delocalized non-specific interaction surface. 2014, Pubmed
Duarri, Molecular pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts: mutations in MLC1 cause folding defects. 2008, Pubmed , Xenbase
Durisic, Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. 2014, Pubmed , Xenbase
Durisic, Stoichiometry of the human glycine receptor revealed by direct subunit counting. 2012, Pubmed , Xenbase
Eertmoed, Transient expression of heteromeric ion channels. 1998, Pubmed
Engelhardt, Effects on channel properties and induction of cell death induced by c-terminal truncations of pannexin1 depend on domain length. 2015, Pubmed , Xenbase
Fields, Nonsynaptic communication through ATP release from volume-activated anion channels in axons. 2010, Pubmed
Frantseva, Specific gap junctions enhance the neuronal vulnerability to brain traumatic injury. 2002, Pubmed
Gandhi, The voltage-clamp fluorometry technique. 2008, Pubmed , Xenbase
Hammer, A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity. 2015, Pubmed , Xenbase
Hines, Inferring subunit stoichiometry from single molecule photobleaching. 2013, Pubmed
Hoffmann, Physiology of cell volume regulation in vertebrates. 2009, Pubmed
Hyzinski-García, LRRC8A protein is indispensable for swelling-activated and ATP-induced release of excitatory amino acids in rat astrocytes. 2014, Pubmed
Jackson, Single channel properties of a volume sensitive anion channel: lessons from noise analysis. 1996, Pubmed
Jentsch, VRAC: molecular identification as LRRC8 heteromers with differential functions. 2016, Pubmed
Manolopoulos, Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells. 1997, Pubmed
McGuire, Automating single subunit counting of membrane proteins in mammalian cells. 2012, Pubmed , Xenbase
Michalski, Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop. 2016, Pubmed
Minieri, The inhibitor of volume-regulated anion channels DCPIB activates TREK potassium channels in cultured astrocytes. 2013, Pubmed
Minieri, Intracellular Na(+) inhibits volume-regulated anion channel in rat cortical astrocytes. 2015, Pubmed
Mongin, Volume-regulated anion channel--a frenemy within the brain. 2016, Pubmed
Nilius, Properties of volume-regulated anion channels in mammalian cells. 1997, Pubmed
Pasantes-Morales, Brain volume regulation: osmolytes and aquaporin perspectives. 2010, Pubmed
Pedersen, The identification of a volume-regulated anion channel: an amazing Odyssey. 2015, Pubmed
Pedersen, Biophysics and Physiology of the Volume-Regulated Anion Channel (VRAC)/Volume-Sensitive Outwardly Rectifying Anion Channel (VSOR). 2016, Pubmed
Penuela, The biochemistry and function of pannexin channels. 2013, Pubmed
Planells-Cases, Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt-based anti-cancer drugs. 2015, Pubmed
Preston, Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. 1992, Pubmed , Xenbase
Qiu, SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel. 2014, Pubmed
Sandilos, Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region. 2012, Pubmed
Sandilos, Physiological mechanisms for the modulation of pannexin 1 channel activity. 2012, Pubmed
Simonin, The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2. 2015, Pubmed , Xenbase
Smits, LRRC8 extracellular domain is composed of 17 leucine-rich repeats. 2004, Pubmed
Stauber, The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology. 2015, Pubmed
Stühmer, Electrophysiologic recordings from Xenopus oocytes. 1998, Pubmed , Xenbase
Syeda, LRRC8 Proteins Form Volume-Regulated Anion Channels that Sense Ionic Strength. 2016, Pubmed
Ulbrich, Subunit counting in membrane-bound proteins. 2007, Pubmed , Xenbase
Ullrich, Inactivation and Anion Selectivity of Volume-regulated Anion Channels (VRACs) Depend on C-terminal Residues of the First Extracellular Loop. 2016, Pubmed
Voets, The chloride current induced by expression of the protein pICln in Xenopus oocytes differs from the endogenous volume-sensitive chloride current. 1996, Pubmed , Xenbase
Voets, Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels. 1999, Pubmed
Voss, Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. 2014, Pubmed
Yahara, Amino acid residues involved in the substrate specificity of TauT/SLC6A6 for taurine and γ-aminobutyric acid. 2014, Pubmed , Xenbase