Gaitán-Peñas H et al. (2016), Investigation of LRRC8-Mediated Volume-Regulate...

XB-ART-52563

Biophys J 2016 Oct 04;1117:1429-1443. doi: 10.1016/j.bpj.2016.08.030.

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

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

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Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

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Species referenced: Xenopus
Genes referenced: lrrc8a mapt nbl1 sri uqcc6

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	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.

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