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. 2016 Oct;4(19):e12940.
doi: 10.14814/phy2.12940.

Leucine-rich Repeat Containing Protein LRRC8A Is Essential for Swelling-Activated Cl- Currents and Embryonic Development in Zebrafish

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Free PMC article

Leucine-rich Repeat Containing Protein LRRC8A Is Essential for Swelling-Activated Cl- Currents and Embryonic Development in Zebrafish

Toshiki Yamada et al. Physiol Rep. .
Free PMC article

Abstract

A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl(-) and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8(-/-) cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl(-) current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa VRAC activity is fully reconstituted in LRRC8(-/-) human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.

Keywords: Cell swelling; cell volume regulation; chloride channel; organic anion channel.

Figures

Figure 1
Figure 1
Current‐to‐voltage relationships of swelling‐activated anion currents in various cell types from adult zebrafish. Plots show basal current and peak current observed 6 min after induction of swelling by exposure of cells to a 250 mOsm. Currents were recorded from dissociated ventricular myocytes and kidney cells and unidentified blood cells. Whole cell currents were evoked by ramping membrane potential from −100 to +100 mV over 200 msec. Cells were held at 0 mV for 5 sec between voltage ramps. Values are means ± SE (n = 4–7).
Figure 2
Figure 2
Characteristics of swelling‐activated anion currents in zebrafish embryo cells. (A) Time course of swelling‐induced activation and shrinkage‐induced inactivation of whole cell Cl currents. Cells were swollen by exposure to a 250 mOsm bath solution and then shrunken by returning to an isotonic 300 mOsm bath. Currents were evoked by ramping membrane potential from −100 to +100 mV over 200 msec. Cells were held at 0 mV for 5 sec between voltage ramps. For each cell, basal anion currents measured during the first 30 sec after obtaining whole cell access were averaged and subtracted from all current recordings. Values are means ± SE (n = 3). (B) Current‐to‐voltage relationships of basal current, peak current observed 2.5 min after induction of swelling by exposure of cells to a 250 mOsm bath solution and current measured 2 min after shrinking swollen cells by returning them to isotonic 300 mOsm bath solution. Currents were evoked as described in (A). Values are means ± SE (n = 3). (C) Representative whole cell current traces of basal and swelling‐activated anion currents. Currents were evoked by voltage clamping cells from −120 to +120 mV for 1 sec in 30 mV increments from a holding potential of 0 mV. Each test pulse was followed by a 2‐sec recovery period at 0 mV. (D) Effect of 10 μmol/L DCPIB on swelling‐activated whole cell anion current. DCPIB was added to the bath 2 min after cell swelling was induced by exposure to a 250 mOsm bath solution. Inhibitory effect of DCPIB was complete within 2 min after adding the drug to the bath solution. Currents were evoked as described in (A). Values are means ± SE (n = 3).
Figure 3
Figure 3
Alignment of predicted zebrafish Lrrc8aa and human and mouse LRRC8A amino acid sequences. Predicted transmembrane (TM) domains are shown in red. Green boxes outline 17 predicted (http://www.uniprot.org/uniprot/Q8IWT6) leucine‐rich repeat domains located on the cytoplasmic carboxy‐terminus.
Figure 4
Figure 4
Inhibition of embryo cell swelling activated Cl currents with translation (ATG) and splice blocking (Splicing) morpholino (MO) knockdown of lrrc8aa. (A, B) Time course and current‐to‐voltage relationships of swelling induced Cl currents in embryo cells injected with control or ATG MO. For each cell in (A), basal anion currents measured during the first 30 sec after obtaining whole cell access were averaged and subtracted from all current recordings. Values are means ± SE (n = 6–8). P < 0.0004 compared to control. *P < 0.003 compared to control at all voltages. (C) Current‐to‐voltage relationships of swelling‐activated Cl currents in embryo cells injected with control or Splicing MO. Values are means ± SE (n = 5–7). **P < 0.03 for all voltages except +20 mV. Cell swelling was induced in all experiments by exposure to a 250 mOsm bath solution and currents were evoked by ramping membrane potential from −100 to +100 mV over 200 msec. Cells were held at 0 mV for 5 sec between voltage ramps.
Figure 5
Figure 5
Expression of LRRC8 cDNAs in LRRC8 −/− HCT116 cells. (A) Time course of swelling‐induced activation and shrinkage‐induced inactivation of whole cell Cl currents. Cells were swollen by exposure to a 250 mOsm bath solution and then shrunken by returning to a hypertonic 400 mOsm bath. Currents were evoked by ramping membrane potential from −100 to +100 mV over 200 msec. Cells were held at 0 mV for 5 sec between voltage ramps. Values are means ± SE (n = 3–8). (B) Current‐to‐voltage relationships of basal and peak swelling‐activated Cl currents. Values are means ± SE (n = 4). (C) Representative whole cell current traces of swelling‐activated anion currents. Currents in (B) and (C) were evoked by voltage clamping cells from −140 to +140 mV for 2 sec in 20 mV increments from a holding potential of 0 mV. Each test pulse was followed by a 3‐sec recovery period at 0 mV.
Figure 6
Figure 6
Expression of lrrc8aa and effect of lrrc8aa knockdown during zebrafish embryogenesis. (A) Relative expression of lrrc8aa during various stages of embryonic development. Values are means ± SE (n = 3). *P < 0.002 compared to 1000‐cell stage. (B) Brightfield micrographs of 48 hpf larvae developing from one‐cell stage embryos injected with 1 mmol/L control MO or 0.1–1 mmol/L lrrc8aa ATG MO. Asterisk and arrows show larvae with moderate and severe phenoptypes, respectively. (C) Penetrance of larval phenotypes. Values are means ± SE (n = 3 independent experiments with 185–232 animals).

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