FASEB J. 2009 Nov;23(11):3829-42. Epub 2009 Jul 13.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species.

Lazrak A, Iles KE, Liu G, Noah DL, Noah JW, Matalon S.

Source

Department of Anesthesiology, Schools of Medicine and Public Health, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Abstract

The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride-sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride-sensitive currents in both Xenopus oocytes, injected with human alpha-, beta-, and gamma-ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10-aa region within the M2 C terminus prevented 70% of this effect. The M2 ENaC down-regulation occurred at normal pH and was prevented by MG-132, a proteasome and lysosome inhibitor. M2 had no effect on Liddle ENaCs, which have decreased affinity for Nedd4-2. H441 and A549 cells transfected with M2 showed higher levels of reactive oxygen species, as shown by the activation of redox-sensitive dyes. Pretreatment with glutathione ester, which increases intracellular reduced thiol concentrations, or protein kinase C (PKC) inhibitors prevented the deleterious effects of M2 on ENaCs. The data suggest that M2 protein increases steady-state concentrations of reactive oxygen intermediates that simulate PKC and decrease ENaCs by enhancing endocytosis and its subsequent destruction by the proteasome. These novel findings suggest a mechanism for the influenza-induced rhinorrhea and life-threatening alveolar edema in humans.

PMID:: 19596899; [PubMed - indexed for MEDLINE]
PMCID:: PMC2775009

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Figure 2.

A) M2 inhibits ENaC. Xenopus oocytes were microinjected with α-, β-, and γ-hENaC cRNAs (8.4 ng each). Twenty-four hours later, they were microinjected with either Udorn M2 cRNA (25 ng) or an equal volume of RNA-free sterile water. Whole-cell I_ENaC was measured 24 h after M2 injection. B) M1 does not inhibit ENaCs. Xenopus oocytes were microinjected with either α-, β-, and γ-hENaC cRNAs (8.4 ng each) plus sterile water or α-, β-, and γ-hENaC cRNAs plus Udorn M1 cRNA (25 ng). Whole-cell currents were recorded 48 h later. Values are means ± se; n = number of oocytes.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 4.

M2 and ENaC do not colocalize in transfected H441 cells. H411 cells were seeded on glass coverslips 24 h before transfection with M2 plasmids. Twenty hours later they were fixed, stained, and imaged with confocal microscopy, as mentioned in Materials and Methods. Images shown are at the level of the plasma membranes. A) Control H441 cells expressing endogenous ENaC were immunostained with a primary antibody to α-ENaC (rabbit) followed by a secondary antibody (donkey anti-rabbit) coupled to Texas Red (red). B) Same image as A with cell nucleus staining. C) H441 cells transfected with M2 cDNA plasmid showing α-ENaCs (red). D) Same cells as in C, immunostained with a primary antibody to M2 (mouse) followed by a secondary antibody (goat anti-mouse) coupled to Alexa Fluor 488 (green). E, F) Merged images of C and D without (E) and with (F) nucleus staining (blue). Note that there is no immunocolocalization of α-ENaC and M2 protein. Typical images were reproduced 9 times with the same results.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 6.

Transfection of A549 cells with M2 increases intracellular levels of ROS. A549 cells were transfected with a RFP-containing plasmid (A–C) or a plasmid containing both RFP and M2 (D–F), as described in Materials and Methods. Forty-eight hours later, the A549 cells were incubated with DCF-DA following the manufacturer’s instructions. Nuclei were counterstained with Hoechst dye (blue). A, D) Red fluorescence, indicating cells transfected with plasmids. B, E) Green fluorescence, indicating increased production of reactive species. C, F) Merged images of red and green fluorescence. Note yellow-orange color in cells transfected with RFP-M2 plasmids (F) but not with RFP alone (C). Typical records reproduced repeatedly with 6 different preparations.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 8.

Mitigation of M2 ENaC down-regulation by reactive species scavengers and PKC inhibitors. I_ENaC at −100 mV. Values are means ± se; numbers of measurements are shown next to each bar. A) Oocytes microinjected with α-, β-, and γ-hENaC or ENaC + M2 cRNAs; 500 μM GSH ester was added in the medium, and the solution was changed every 12 h. B) A549 cells transfected with M2 plasmids containing either GFP alone or M2 and GFP genes; 500 μM GSH ester was added in the medium, as described above. C, D) H441 cells transfected with plasmids containing either GFP or GFP plus M2 genes; Gö6796 (250 nM; a specific inhibitor of PKCα/β₁ isoforms) (C) or pseudosubstrate inhibitor, myristoylated (1 μM; a specific PKCζ inhibitor) (D) was added into the medium 24 h later for 24 h at which time I_ENaC was recorded. *P < 0.01 compared with the corresponding control (either ENaCs for oocytes or GFP for cells). ^#P < 0.01 vs. value to immediate left; Student’s t test.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 1.

M2 inhibits amiloride-sensitive Na⁺ currents at neutral pH of Xenopus oocytes and H441 cells. A, B) I-V relationships of I_M2 recorded from Xenopus oocytes 48 h after microinjection with Udorn (A) and WSN (B) M2 cRNAs (25 ng), using the 2-electrode voltage-clamp technique, at extracellular pH 7.6 or 5.5, as described in Materials and Methods. I-V relationships were obtained before and after perfusion with amantadine (100 μM). C) I_M2 in H441 cells transfected with a plasmid containing the Udorn M2 and GFP genes. Currents were measured in H441 expressing green fluorescence 48 h post-transfection at bath pH values of either 7.4 or 5.5; the proton current was totally inhibited by 100 μM amantadine. D) I-V relationships of I_ENaC from oocytes coinjected with M2 (25 ng) and α-, β-, and γ-hENaC cRNAs (8.4 ng each) 48 h after injection. Difference currents before and after perfusion with amiloride (10 μM) at a given potential are shown. E) Total currents across H441 cells transfected with plasmids containing either GFP alone or GFP-M2 before and after addition of amiloride (10 μM) in the bath solution (pH 7.4). Cells were held at −40 mV, and membrane potentials were changed from −100 to +100 mV in 10-mV increments for 500 ms. F) I-V relationships of I_ENaC of nontransfected H441 cells and cells transfected with plasmids containing either GFP or GFP + M2. In all cases (except E) values are means ± se; n = number of oocytes or H441 cells.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 3.

M2 reduces single-channel ENaC activity and P_o. A, B)Typical records (A) and corresponding amplitude histograms (B) of single channels from cell-attached patches of Xenopus oocytes 48 h after microinjection with α-, β-, and γ-hENaCs alone or ENaCs + M2. Amplitude histograms were constructed from records of at least 10 min. C) Western blots of total [whole-cell extract (WCE)] and membrane α- and β-hENaC levels from Xenopus oocytes microinjected with either ENaCs or ENaCs + M2. GAPDH levels indicate uniform loading of lanes. D) Western blots of γ-ENaC in biotinylated proteins from oocytes microinjected with either α-, β-, and γ-hENaCs or ENaCs + M2. Biotinylated proteins were precipitated with streptavidin-agarose, separated by SDS-PAGE, followed by Western blotting with anti-γ-ENaC antibodies, as described in Materials and Methods. Similar levels of nonspecific bands indicate uniform loading of each lane. Typical records were repeated 3 times with identical results. We were unable to detect α- and β-hENaC in biotinylated proteins, despite the fact that we could detect them in total membrane fractions (C).

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 5.

A) Identification of regions of M2 responsible for decreased ENaC activity. Amino acid sequence of the A/Udorn/1972 M2 protein and the points of truncation for each construct are shown. The 3 protein domains are indicated above the sequence. B) Xenopus oocytes were coinjected with α-, β-, and γ-hENaC cRNAs and sterile water or with α-, β-, and γ-hENaC cRNAs plus either the full-length M2 or truncated M2 cRNAs. Forty-eight hours later, I_ENaC (left panel) and I_M2 (right panel) were recorded. Values were obtained at a membrane potential of −100 mV and are means ± 1 se; n = ≥21. *P < 0.01 vs. control; ^#P < 0.01 vs. corresponding ENaC or M2. B) MG-132 reverses the M2 effect on ENaCs. Oocytes were injected with α-, β-, and γ-hENaC or α-, β-, and γ-hENaC and M2 cRNAs. In some cases, MG-132 (40 nM) was added in the medium immediately after injection. Whole-cell currents were recorded 48 h later, and I_ENaC was calculated as described in Materials and Methods. Values are means ± se; n = number of measurements. C) M2 does not down-regulate ENaCs with Liddle mutations. Xenopus oocytes were injected with α-, β-, and γ-ENaC cRNAs with the Liddle mutation alone or Liddle ENaC and M2 cRNAs. I_ENaC was recorded as described in Materials and Methods. Values are means ± 1 se; n = number of measurements.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Figure 7.

Transfection of A549 cells with M2 increases mitochondrial levels of reactive species. A549 cells were transfected with a GFP-containing plasmid (A–C) or a plasmid containing both GFP and M2 (D–F). Forty-eight hours later, the A549 cells were incubated with the MitoSOX reagent following the manufacturer’s instructions. Nuclei were counterstained with Hoechst (blue). A, D) Green fluorescence, indicating cells transfected with plasmids. B, E) Red fluorescence, indicating increased production of reactive species, most likely by mitochondria. C, F) Merged images of red and green fluorescence. Note yellow-orange color in cells transfected with RFP-M2 plasmids (F) but not with RFP alone (C). Typical records were reproduced repeatedly with 6 different preparations.

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

FASEB J. FASEB J;23(11):3829-3842.

Publication Types, MeSH Terms, Substances, Grant Support

Publication Types

Research Support, N.I.H., Extramural

MeSH Terms

Amiloride/pharmacology
Animals
Cells, Cultured
Epithelial Sodium Channel/antagonists & inhibitors*
Humans
Oocytes/metabolism
Protein Kinase C/metabolism
Reactive Oxygen Species/metabolism*
Transfection
Viral Matrix Proteins/physiology*
Xenopus laevis

Substances

Epithelial Sodium Channel
M2 protein, Influenza A virus
Reactive Oxygen Species
Viral Matrix Proteins
Amiloride
Protein Kinase C

Grant Support

1U54ES017218/ES/NIEHS NIH HHS/United States
5P30DK072482/DK/NIDDK NIH HHS/United States
5U01ES015676/ES/NIEHS NIH HHS/United States
AI071393/AI/NIAID NIH HHS/United States
HL031197/HL/NHLBI NIH HHS/United States

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Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species.

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