Grant GJ et al. (2020), Oxidized Glutathione Increases Delta-Subunit Ex...

XB-ART-57175

Emed Res 2020 Jan 01;2.

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Oxidized Glutathione Increases Delta-Subunit Expressing Epithelial Sodium Channel Activity in Xenopus laevis Oocytes.

Grant GJ , Coca C , Zhao XM , Helms MN .

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Epithelial sodium channels (ENaC) are heterotrimeric structures, made up of α, β, and γ subunits, and play an important role in maintaining fluid homeostasis. When δ-ENaC subunits are expressed in place of (or in addition to) the α-ENaC subunit alongside β- and γ- subunits, fundamental changes in the biophysical properties of ENaC can be observed. Using human ENaC cRNA constructs and the Xenopas laevis oocyte expression system, we show that oxidized glutathione (GSSG) differently effects αβγ-ENaC and αβγ-ENaC current. GSSG (400 μM) significantly decreased normalized whole cell current in oocytes expressing αβγ-ENaC, and conversely increased whole cell current in δ1βγ-ENaC and δ2βγ-ENaC expressing oocytes. GSSG treatment increased current in oocytes expressing all four subunits. Western blot and PCR analysis show that human small airway epithelial cells (hSAEC) express canonical αβγ-subunits alongside δ-ENaC subunits. Differences in single channel responses to GSSG in hSAECs indicate that airway epithelia redox sensitivity may depend on whether δ- or α- subunits assemble in the membrane. In silico analysis predict that six Cys amino acids in the δ-ENaC extracellular loop, and a single Cys in the N-terminal domain, are susceptible to post-translational modification by GSSG. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of oxidized glutathione and δ-ENaC in lung disorders.

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Species referenced: Xenopus laevis

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	Figure 2. Glutathione disulfide (400 μM) increases δ1βγ-ENaC whole cell current A) Representative amiloride-sensitive δ1βγ-ENaC whole cell current; arrow indicates 0 current. Test pulses between −80 mV to +40 mV shown in 20 mV increments. B) GSSG perfusion increases δ1βγ-ENaC whole cell current and returns to basal levels following wash out (WO) in the same cell. C) Representative time course trace of GSSG activation of δ1βγ-ENaC; −60 mV holding potential. D) Normalized current of CTR, GSSG, and WO treated δ1βγ-ENaC expressing oocytes at −80 mV, wherein δ1βγ-ENaC current increased from −4.3 ± 0.50 μA to −6.0 ± 1.16 μA; n=12 independent observations from 3 different oocyte preparations, *=p<0.05 using Paired t-test comparisons.
	Figure 3. Glutathione disulfide (400 μM) increases δ2βγ-ENaC whole cell current A) Representative amiloride-sensitive δ2βγ-ENaC whole cell current; arrow indicates 0 current. Test pulses between −80 to +60 mV shown in 20 mV increments. B) GSSG perfusion increases δ2βγ-ENaC current and returns to basal levels following washout (WO). C) Representative time course of GSSG induced activation of δ2βγ-ENaC whole cell current plotted at −60 mV holding potential. D) Normalized whole cell current of CTR and GSSG treated δ1βγ-ENaC expressing oocytes at −80 mV, wherein GSSG increased current from δ2βγ-ENaC expressing oocytes from −3.9 ± 0.16 μA to −5.0 ± 0.50 μA; n=13 independent observations from 3 different oocyte preparations, *=p<0.05 using Paired t-test comparisons.
	Figure 4. Human small airway epithelial cells express α, β, γ and δ-ENaC subunits A) Conventional and nested PCR analysis of α-, β, γ- and δ-ENaC transcript expression in hSAEC on 1.2% agarose gel; arrow indicates 500 base pair (bp) molecular size indicator. B) Quantitative real time PCR determination of threshold cycles (ΔCt) for α-, β-, γ- and δ-ENaC transcript expression in hSAEC. C) Immuno-histochemical detection of α-, β-, γ- and δ-ENaC protein in hSAECs as indicated. Nuclei were labeled with DAPI. No primary antibody was added to the control (CTR) panel to show that positive signals are not due to non-specific binding of the secondary goat anti-rabbit IgG-Alexa Fluor 488 antibody. Fluorescence signal threshold defined using CTR settings and remained consistent for all subunits analyzed. D,E) Representative cell-attached single channel analysis of hSAEC before (red trace) and following 400 μM GSSG application to extracellular bath. A portion of GSSG-induced increase in activity is enlarged to show single channel detail; downward deflections from arrow indicates channel opening. Histogram in the inlay shows freguency of channels in closed (0 pA) or open state. GSSG increased activity as shown in panel D, but decreased activity in panel E. F) Distinct and varied response of hSAECs to GSSG; 44% of single channel recordings showed a GSSG-induced decrease in ENaC Po, and 56% of the recordings showed a GSSG-induced increase in ENaC Po; n=16 independent patch-clamp recordings from hSAECs.
	Figure 5. Glutathione disulfide (400 μM) increases αβγδ-ENaC whole cell current A) GSSG perfusion increases αβγδ-ENaC cell current, which returns to baseline levels following wash out (WO); arrow indicates 0 current. Voltage steps between −80 to +60 mV shown in 20 mV increments. B) Representative time course of GSSG induced activation of αβγδ-ENaC whole cell current plotted at 60 mV holding potential. C) Normalized current of CTR and GSSG treated aPyb-ENaC expressing oocytes at −80 mV, wherein GSSG decreased current from −4.5 ± 0.80 pA to −5.58 ± 0.7 μA; n=13 independent observations from 2 different oocyte preparations, *=p<0.05 using Paired t-test comparisons. GSSG washout returned whole cell current to baseline levels in each observation.
	Figure 6. In silico analysis of δ-ENaC S-glutathionylation A) Amino acid sequence of N-terminal, transmembrane domains 1 and 2, extracellular loop, and C-terminal domains of δ1 and δ2 ENaC subunits with predicted protein-SSG site highlighted. B) Illustration showing site of predicted Cys glutathionylation in α, δ1, and δ2 ENaC isoforms. C-E) 10 μM NEM pretreatment attenuates GSSG-mediated changes in ENaC whole cell current, αβγ, N=11, δ1βγ, N=11, δ2βγ N=8 from 3 different batches of oocytes. All whole cell currents were normalized to current amplitudes at −60 mV prior to NEM treatment; *=p<0.05 using Paired t-test comparisons.

References [+] :

Agrawal, The Epithelial Sodium Channel Is a Modifier of the Long-Term Nonprogressive Phenotype Associated with F508del CFTR Mutations. 2017, Pubmed, Xenbase