Biophysical Journal, Volume 111

Supplemental Information

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in

Xenopus Oocytes

Héctor Gaitán-Peñas, Antonella Gradogna, Lara Laparra-Cuervo, Carles Solsona, VictorFernández-Dueñas, Alejandro Barrallo-Gimeno, Francisco Ciruela, MelikeLakadamyali, Michael Pusch, and Raúl Estévez

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Supplementary Material

Investigation of LRRC8-mediated volume-regulated anion currents in Xenopus oocytes

reveals novel properties

H. Gaitán-Peñas, A. Gradogna, L. Laparra-Cuervo, C. Solsona, V. Fernández-Dueñas, A.

Barrallo-Gimeno, F. Ciruela, M. Lakadamyali, M. Pusch, R. Estévez

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Supplementary Figure 1. Custom device for luminescence measurements.

ATP induced luminescence from luciferin-luciferase mixture was measured using a

home-made device in which a chamber containing an assay tube can be exposed to a

photomultiplier (PM). The set up was inspired by a device described by Israel and

Lesbats (1). The PM is fed with a high voltage power supply delivering (HT) and

generates a signal which increases upon light emission. The chamber (A), made in black

anodized aluminium and connected to the PM, consists of three parts: A lid, a cubic block

and a cylinder. The lid isolates the inner part of the chamber from any external source of

light. The cubic block, in black, has a window that feeds light to the PM. The cubic block

has a cylindrical cavity with a tightly fitted but rotatable cylinder (1, in grey), made as well

with black anodized aluminium. The cylinder is coupled to the lid in a manner in which it

can be turned on right or left by rotating the lid. A’ represents an air view of the top of

chamber; the white dashed line represents the perimeter of the lid. The cylinder bears a

cavity with the size of a hemolysis tube. The lateral part of this cavity contains a window

at the same height as the window in the cubic block, allowing optical contact between the

hemolysis tube and the PM. For ATP release experiments, Luciferin-luciferase mixture

was first added to the tube without oocytes, and the background light signal due to

spontaneous emission was recorded in position B, B’. After this, oocytes were added in

position A, A’, the tube was rotated back to position B, B’, and light emission was

continuously monitored. Drugs or other solutions were added in an analogous manner.

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Supplementary Figure 2. Current recovery of hypotonically activated 8A/8E and 8A-VFP/8E-mCherry channels.

Effect of osmolarity changes on 8A/8E (A) and 8A-VFP/8E-mCherry (B). Currents are

plotted as function of the time. Colors correspond to the different solutions applied during

the experiment: black: 200 mOsm (“Iso” solution), cyano: 120 mOsm (“Hypo” solution),

red: 100 µM CBX. After activation in hypotonic medium 8A/8E (panel A) as well as 8A-

VFP/8E-mCherry (panel B) expressing oocytes currents at 60 mV slowly returned to

almost baseline values. CBX was applied to test for the specificity of the activated

currents.

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Supplementary Figure 3. Functional expression of untagged LRRC8 heteromers in

Xenopus oocytes. (A-B) Mean values of currents at 60 mV recorded from oocytes in

which 8A was co-injected with 8B (A) and 8C (B) in “Iso” solution, and after 5 min and 10

min in “Hypo” solution. (n ≥ 4). (C) Functional expression of 8A/8D. Voltage clamp traces

from a 8A/8D injected oocyte evoked by the “IV-pulse protocol” in “Iso” solution (top, left)

and after 5 min perfusion of “Hypo” solution (top, right). Mean current values in “Iso”

solution, and after 5 min and 10 min in “Hypo” solution (bottom) (n ≥ 3). The dashed line

in bottom panels A-D indicates an arbitrary threshold of current expression corresponding

to 0.5 µA. Error bars indicate SD.

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Supplementary Figure 4. Analysis of currents induced by the expression of 8E-

mCherry in Xenopus oocytes. Time course between 2 and 4 days after oocyte injection

of the mean values of currents at 60 mV for un-injected oocytes (n≥7), oocytes injected

with 8E-mCherry (n≥18) and oocytes injected with 8A-VFP/8E-mCherry (n≥7). Data are

from at least 2 different batches of oocytes and indicate the mean ± s.e.m.

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Supplementary Figure 5. Activation of LRRC8 channel by various tags. VFP,

mCherry, 3xHA or 3xflag tags were C-terminally fused to LRRC8A and LRRC8E

constructs and co-injected as indicated. Currents were measured at 80 mV after 10 min

incubation in hypotonic ND48 medium. Statistical comparison was performed with

currents of untagged 8A/8E expressing oocytes. ***: p<0.001, **: p<0.01. Errors bars

indicate s.e.m. (n>=6 oocytes for each group; qualitatively similar results were obtained

in another batch).

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Supplementary Figure 6. Western blot analysis of the expression of fluorescently

tagged LRRC8 proteins.

Extracts from un-injected oocytes or oocytes injected with 8A-VFP alone, 8E-mCherry

alone or 8A-VFP plus 8B/C/D/E-mCherry were obtained. Western blots against

antibodies for GFP or Cherry were performed, using -tubulin as a loading control. Two

independent WB experiments gave similar results. The expected size of the fluorescently

tagged proteins is between 119 and 127 kDa. However, the banding pattern suggests

altered post-translational modifications such as glycosylation, phosphorylation or partially

translated products. It has been recently shown (2) that PNGaseF treatment of LRRC8A,

but not of LRRC8A-N66A/N83A with disrupted glycosylation sites, decreased LRRC8A

size in Western blots, indicating that LRRC8A is glycosylated at N66 and N83.

Furthermore, as LRRC8A-N66A/N83A also had a banding pattern, LRRC8A may also

contain other post-translational modifications such as phosphorylation. According to

Uniprot, all LRRC8 proteins contain putative sites of phosphorylation and glycosylation.

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Supplementary Figure 7. Typical data used for the determination of ion selectivity.

Panels A and B show data for 8A-VFP/8E-mCherry. In panel A typical “tail current” traces

are shown in the Cl- solution, in Glutamate, and after wash with the Cl- solution. In panel

B typical I-V curves are shown for the indicated ions (data are from different oocytes

because not all ions could be tested in a single oocyte). Solid lines are fits of a parabola

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to the 4 points closest to the reversal potential, which was used to extract the

experimental reversal potential. Panel C shows similar data for 8A-VFP/8D-mCherry.

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Supplementary Figure 8. Functional and cell-biological investigation of the

variability of LRRC8 stoichiometry. (A) TIRF image showing LRRC8E-VFP and

LRRC8D-mCherry oligomers on the Xenopus oocyte membrane in the presence of

untagged LRRC8A and a merge of the two fluorescent channels. Arrowheads point to

overlapping LRRC8E and LRRC8D spots. (B) Example VFP photobleaching traces of

8A-VFP/8E-mCherry expressing oocytes with 1, 2, 4, 5, 6 or >6 bleaching steps. (C)

Normalized currents at 60 mV from oocytes injected with 8A-VFP/8E-mCherry (left) or

8A-mCherry/8E-VFP (right) using either the same amount of each (2.5 ng 8A, 2.5 ng 8E)

or an increased amount of the 8A RNA (3.75 ng 8A, 1.25 ng 8E). Data are the mean from

three independent experiments and indicate the mean ± s.e.m. * p < 0.05, ** p < 0.01, ***

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p < 0.001. (D) Distribution of the percentage of counted photobleaching steps (n=53)

from LRRC8A-VFP oligomers in complex with LRRC8E-mCherry oligomers at the 3:1

RNA ratio.

Supplementary references

1. Israel, M., and B. Lesbats. 1981. Chemiluminescent determination of acetylcholine, and continuous detection of its release from torpedo electric organ synapses and synaptosomes. Neurochem. Int. 3:81-90.

2. Voss, F. K., F. Ullrich, J. Munch, K. Lazarow, D. Lutter, N. Mah, M. A. Andrade-Navarro, J. P. von Kries, T. Stauber, and T. J. Jentsch. 2014. Identification of LRRC8 heteromers as an essential component of the volume-regulated anion channel VRAC. Science 344:634-638.