موجز عن البحث:
The hERG potassium channel is critical for
repolarisation of the cardiac action potential. Reduced expression of hERG at
the plasma membrane, whether caused by hereditary mutations or drugs, results
in long QT syndrome and increases the risk of ventricular arrhythmias. Thus,
it is of fundamental importance to understand how the density of this channel
at the plasma membrane is regulated. We used antibodies to an extracellular
native or engineered epitope, in conjunction with immunofluorescence and
ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells
and validated the findings in rat neonatal cardiac myocytes. The data reveal
that this channel undergoes rapid internalisation, which is inhibited by
neither dynasore, an inhibitor of dynamin, nor a dominant negative construct
of Rab5a, into endosomes that are largely devoid of the transferrin receptor.
These results support a clathrin-independent mechanism of endocytosis and
exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In
agreement, internalised hERG displayed marked overlap with
glycosylphosphatidylinositolanchored GFP, a clathrin-independent cargo.
Endocytosis was significantly affected by cholesterol extraction with
methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative
Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes
clathrin-independent endocytosis via a mechanism involving Arf6.
stress plays a central role in the pathogenesis of atherosclerosis. By
increasing the production of reactive oxygen species, such as H2O2,
oxidative stress causes apoptosis of endothelial cells. Mechanisms by which H2O2
leads to apoptosis, however, are controversial. One study reported that Zn2+ released from H2O2 oxidation of
metallothioneins is the cause of cell death , while the other suggests that H2O2
activates the TRPM2 channel, resulting in Ca2+ influx and
cell death .
address the controversy, we have examined the effect of H2O2
(1 mM, 6h) on human umbilical vascular endothelial cells using live cell
imaging. We monitored changes in the intracellular distribution of free Ca2+
and Zn2+ using Fluo-4 and FluoZin-3 respectively. We
stained lysosomes and mitochondria with LysoTracker and MitoTracker
respectively, and dead cells with propidium iodide. Data recorded from N
number of cells and n number of independent experiments are expressed as
mean±SEM. P value of <0.05
(Student’s t-test) was considered
found that both Ca2+ and Zn2+ are highly enriched in lysosomes.
Mitochondria showed little stain for either ion. H2O2
treatment increased the cytosolic levels of both ions in most cells. In some
cells, however, we found striking redistribution of Zn2+ from lysosomes to mitochondria: There was a decrease in the
number of lysosomes with Zn2+ (untreated: 61±6%,
n/N=3/75; H2O2 treated: 21±1%, n/N=3/49; P<0.01),
with a concomitant rise in mitochondria with Zn2+ (untreated: 11±1%, n/N=3/49; H2O2
treated: 41±4%, n/N=3/34; P<0.01). These effects were found in the
absence of extracellular Zn2+, indicating redistribution of
of TRPM2 with PJ34 and 2-aminoethoxydiphenyl borate (2–APB) reduced the H2O2
induced release of Zn2+ into the cytoplasm (PJ34: 45±2%, n/N=3/43, P<0.01;
2-APB: 76±1 %, n/N=3/38, P<0.05),
as well as its translocation from lysosomes to mitochondria (PJ34: 11±3%,
n/N=3/46, P<0.05; 2-APB: 13±2%, n/N=3/35, P<0.05).
Transfected HA-tagged TRPM2 channels showed co-localisation with the
lysosomal markers, LysoTracker and CD63, suggesting TRPM2 mediates release of
Zn2+ from lysosomes.
determined the relevance of TRPM2 and Zn2+ redistribution to H2O2-induced
endothelial cell death. Blockers of TRPM2 (PJ34 and 2-APB), as well as
chelation of Zn2+ with TPEN (N,N,N’,N’-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine),
completely inhibited H2O2-induced cell death,
indicating TRPM2 mediated changes in the redistribution of Zn2+ contribute to endothelial cell death.
conclusion, we discovered a novel mechanism where H2O2 activation
of TRPM2 causes a redistribution of Zn2+ from lysosomes to mitochondria and
cytoplasm, resulting in endothelial cell death.