Trafficking-deficient hERG K⁺ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER - PubMed (original) (raw)
Trafficking-deficient hERG K⁺ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER
Jennifer L Smith et al. Am J Physiol Cell Physiol. 2011 Jul.
Abstract
The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.
Figures
Fig. 1.
A: representative fluorescent images of cells expressing wild-type (WT) or G601S-human ether-a-go-go related gene (hERG) immunostained with anti-hERG (red, first column) or anti-M6PR (green, second column) studied in control (con) conditions after being treated in 20 μM nocodazole (noc) for 18–24 h. The overlay is shown in the third column. The white arrows in the first column highlight several of the G601S-hERG reticula that form after noc treatment. The fourth column shows a section of the overlay (the white dashed box) in greater detail, and insets show the overlays in greater detail (white dashed box). The scale bars represent 10 μm. B: representative whole cell current traces recorded from cells expressing WT-hERG or G601S-hERG in control or noc-treated conditions measured by prepulsing cells to 50 mV for 3 s followed by a test pulse to −120 mV for 3 s. Mean peak inward current measured during the test pulse from cells in control or noc-treated conditions (*P < 0.05 compared with control) are shown. C: representative Western blot analyses of cells expressing WT-hERG or G601S-hERG in control or noc-treated (20 μM, 18–22 h, n = 5–6) conditions are shown. Top arrow corresponds to a molecular mass of 198 kDa, the middle arrow corresponds to a molecular mass of 125 kDa, and the bottom arrow corresponds to a molecular mass of 90 kDa. The immunoblots were probed with anti-hERG (red) and anti-Na+-K+-ATPase (green). Mean relative density of the 155-hERG band for each condition (*P < 0.05 compared with control) is shown.
Fig. 2.
A: representative infrared signal emissions from a 24-well tissue culture plate. Shown are four wells of confluent cells after anti-hERG and wheat germ agglutinin (WGA) labeling measured from human embryonic kidney (HEK) cells not expressing hERG (background or bg) or cells expressing G601S-hERG in control conditions, after being treated in 10 μM E-4031 for 12 h (e4) or after being treated in 0.2 μM noc for 12 h. B: relative intensities of the anti-hERG immunolabeling normalized to WGA labeling (n = 4, *P < 0.05 compared with bg and #P < 0.05 compared with control).
Fig. 3.
A: representative fluorescent images of cells expressing G601S-hERG immunostained with anti-hERG (red, first column) or anti-M6PR (green, second column) studied after being treated in 20 μM noc for 15 min (row 1), 1 h (row 2), or 2 h (row 3). The third column shows the overlay. The fourth column shows a portion of the overlay (the white dashed box) in greater detail. B: representative fluorescent images of cells expressing G601S-hERG treated with 10 μM podophyllotoxin (pod) or vinblastine (vin) for 2 h (rows 4 and 5, respectively). The white arrows in the first column highlight several of the G601S-hERG reticula that form after drug treatment. The scale bars represent 10 μm.
Fig. 4.
A: representative fluorescent images of cells expressing G601S-hERG immunostained with anti-hERG (red, first column) and different ER markers (green, second column) in control conditions or after being treated in 20 μM noc for 2 h. The second column shows immunostaining for calnexin (first two rows), KDEL proteins (Lys-Asp-Glu-Leu, third and fourth rows), or Sec31 (fifth and sixth rows). The third column shows the overlay. The white arrows in the first column highlight several of the G601S-hERG reticula that form after noc treatment. The scale bars in the third column represent 10 μm. The fourth column shows a portion of the overlays (the white dashed box, third column) in more detail, and insets show the overlays in even greater detail (the white dashed box, fourth column). B: corresponding mean Costes' Pearson's coefficient (PC) for the colocalization of G601S-hERG and the different endoplasmic reticulum (ER) markers in control conditions (calnexin, n = 8 fluorescent images, KDEL proteins, n = 10, Sec31, n = 6) or after noc treatment (calnexin, n = 8, KDEL proteins, n = 10, Sec31, n = 8) (*P < 0.05 compared with control).
Fig. 5.
A: representative fluorescent images of cells immunostained with anti-Sec16a (red, first column) and ERGIC-53 (purple, second column) in control conditions, after being treated in 20 μM noc for 2 h, or after being treated in 20 μM noc and 10 μg/ml brefeldin A (bfa) for 2 h (noc + bfa). The third column shows the overlay. The scale bars in the third column represent 10 μm. The fourth column shows a portion of the overlays (the white dashed box, third column) in more detail, and insets show the overlays in even greater detail (the white dashed box, fourth column). B: corresponding mean Costes' PC for the colocalization of Sec16a and ERGIC-53 in control cells (n = 9), noc-treated cells (n = 9) or noc + bfa-treated cells (n = 11) (*P < 0.05 compared with control; #P < 0.05 for noc compared with noc + bfa).
Fig. 6.
Representative fluorescent images of cells expressing G601S-hERG immunostained with anti-hERG (red, first column) and ERGIC-53 (green, second column) in control conditions after being treated in 20 μM noc for 2 h, or after being treated in 20 μM noc and 10 μg/ml bfa for 2 h (noc + bfa) in the absence (A)or the presence (B) of 10 μM E-4031. The third column shows the overlay. The white arrows in the first column highlight several of the reticula that form after noc or noc + bfa treatment in the absence of E-4031. The scale bars in the third column represent 10 μm. The fourth column shows a portion of the overlays (the white dashed box, third column) in more detail, and insets show the overlays in even greater detail (the white dashed box, fourth column). C: left shows the mean Costes' PC for the colocalization of G601S-hERG and ERGIC-53 in control cells (n = 12 image), after noc treatment (n = 9), or after treatment in noc + bfa (n = 12) in the absence of E-4031. Right shows the mean Costes' PC for the colocalization of G601S-hERG and ERGIC-53 in control cells (n = 8), after noc treatment (n = 9), or after treatment in noc + bfa in the presence of E-4031 (n = 9) (*P < 0.05 compared with control; #P < 0.05 for indicated comparison, and %P < 0.05 compared with without E-4031).
Fig. 7.
Shown are representative fluorescent images of cells expressing G601S-hERG and tsO45-VSV-G-green fluorescent protein (GFP) immunostained with anti-hERG (red, first column) and ERGIC-53 (purple, third column) in control (A) conditions or after being treated in 20 μM noc and 10 μg/ml bfa for 2 h. B: tsO45-VSV-G-GFP fluorescence is shown in the second column. The fourth column shows the overlay. The scale bars represent 10 μm. Insets show the white dashed box in even greater detail. C: left shows the mean Costes' PC for the colocalization of G601S-hERG and tsO45-VSV-G-GFP in control cells (n = 7) or after treatment in noc + bfa (n = 10). Middle shows the mean Costes' PC for the colocalization of G601S-hERG and ERGIC-53 in control cells (n = 7) or after treatment in noc + bfa (n = 10). Right shows the mean Costes' PC for the colocalization of tsO45-VSV-G-GFP and ERGIC-53 in control cells (n = 7) or after treatment in noc + bfa (n = 10) (*P < 0.05 compared with control).
References
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