Bartlett, Wendy

Booth, Ian R.

Castronovo, Samantha

Conway, Stuart J.

Healy, Jess

Lyngberg, Lisbeth

Miller, Samantha

Pliotas, Christos

Rasmussen, Tim

Roosild, Tarmo P.

Healy, Jessica

University of St. Andrews - Pure

Activation and complex regulation of the Kef potassium efflux system during protection of bacteria against toxic electrophiles

Elsevier - Publisher Connector 

102a Sunday, March 6, 2011interactions significantly affect the VDAC properties [1]. The present study fur-
ther explores these lipid-protein interactions with special emphasis on the effect
of phospholipid headgroups. The VDAC was reconstituted in planar lipid bila-
yer formed from either DOPC or DOPE or a DOPE/DOPC mixture. The main
findings of this study are: (a) DOPE is essential for the channel gating at salt
concentration prevailing in vivo and the voltage-dependence is inhibited
when DOPE is replaced by DOPC (b) in the presence of a DOPC/DOPE mix-
ture, the DOPE molal fraction require to restore the VDAC gating is lower than
that required to change the membrane viscosity, c) the unitary conductance is
not affected by the DOPC or DOPE, (d) both selectivity and voltage-depen-
dence are modulated by the degree of methylation of the DOPE but not the sin-
gle channel conductance and the gating parameters. The high resolution
structure of mammalian VDAC was used as a template to identify putative res-
idues able to interact with PE headgroups. All together these results indicate
that there is a direct interaction between PE and the VDAC and that PC locks
the VDAC in its open state inhibiting transitions of the VDAC to the subcon-
ductance states.
[1] Mlayeh et al. Biophys.J. (2010),doi:10.1016/j.bpj.2010.07.067
551-Pos Board B351
Activation and Complex Regulation of the Kef Potassium Efflux System
During Protection of Bacteria Against Toxic Electrophiles
Tim Rasmussen, Christos Pliotas, Lisbeth Lyngberg, Jess Healy,
Wendy Bartlett, Samantha Miller, Tarmo P. Roosild, Samantha Castronovo,
Stuart J. Conway, Ian R. Booth.
Electrophiles react rapidly with DNA and proteins and are therefore toxic. Bac-
teria protect themselves from the immediate effects of electrophiles by lower-
ing the cytosolic pH before the compounds are detoxified. This is achieved by
the Kef systems, which mediate potassium efflux, in turn coupled to an influx of
protons. We investigated the activation and complex regulation of this system.
When electrophiles enter the cell, the most likely reaction partner is the nucle-
ophile glutathione which is present at high concentration in the cytosol. The
resulting glutathione adducts activate Kef while glutathione itself inhibits.
The kinetics of activation was investigated in vivo using a potassium selective
electrode. Activator and inhibitor bind to the same binding site on the regula-
tory KTN domain of Kef and cause differential structural changes, as shown by
crystal structures of this domain [1]. We characterised the binding and these
structural changes further using fluorescence and EPR spectroscopy in purified
Kef proteins. A second binding site on the KTN domain binds nucleotides.
Furthermore, Kef is regulated by a soluble protein which serves as activator
[2]. We present data that this activator protein also has quinone oxidoreductase
activity. The relationship between this enzymatic activity and its function as an
activator of Kef was dissected using point mutations to disrupt enzyme activity
but preserve structural integrity.
[1] Rossild et al., Structure 17, 893 (2009).
[2] Miller et al., J. Bac.182, 6536 (2000).
552-Pos Board B352
Involvement of Caveolin-1 in Probucol-Reduced hERG Membrane
Expression
Jun Guo, Shetuan Zhang.
The human ether-a-go-go-related gene (hERG) encodes the pore-forming sub-
unit of the rapidly activating delayed rectifier Kþ current (IKr) important for
cardiac repolarization. Dysfunction of hERG channels causes Long QT Syn-
drome (LQTS). While diverse compounds reduce the hERG current (IhERG)
by blocking hERG channels, probucol, a cholesterol-lowering drug that
causes LQTS, reduces IhERG by decreasing hERG channel expression in the
plasma membrane. In the present study, we investigated the mechanisms of
probucol effects on hERG expression. Probucol did not block hERG forward
trafficking; it instead accelerated degradation of mature hERG channels. Im-
munocytochemistry and co-immunoprecipitation (Co-IP) data demonstrated
association between hERG channels and caveolin-1 (Cav-1). In HEK cells
without hERG expression, probucol promoted endogenous Cav-1 degrada-
tion. In hERG-expressing HEK cells, overexpression of Cav-1 enhanced,
whereas knockdown of Cav-1 impeded probucol-induced reduction of
hERG channels. Thus, probucol reduces hERG expression through accelerat-
ing Cav-1 turnover. The effects of probucol on Cav-1 and hERG result from
probucol’s cholesterol-disrupting action, since low density lipoprotein (LDL),
a potent cholesterol carrier, effectively prevented probucol-induced reduction
of IhERG in hERG-HEK cells and of IKr in cultured neonatal rat ventricular
myocytes. In conclusion, our data provide evidence that targeting hERG-
interacting protein, caveolin, represents a novel mechanism for drug-induced
hERG reduction and LQTS. (Supported by the Canadian Institutes of Health
Research)553-Pos Board B353
Involvement of Caveolin-3 in the Endocytic Degradation of the Cell
Surface hERG Channels
Xian Li, Jun Guo, Shetuan Zhang.
The human ether-a-go-go-related gene (hERG) encodes the pore-forming sub-
units of the rapidly activating delayed rectifier potassium channel (IKr) that is
important for cardiac repolarization. We have discovered that hERG channels
rapidly internalize in low extracellular Kþ conditions ([Kþ]o), known clinically
as hypokalemia. In cell culture, this internalization process is driven by
endocytic protein, caveolin-1 (Cav-1), which is an integral player in the caveo-
lae-dependent endocytosis pathway. However, Cav-1 is tissue specific; its
homolog, Caveolin-3 (Cav-3), is the predominant form in muscles and thus
may play a direct role in regulating hERG expression in cardiac myocytes.
To investigate the roles of Cav-3 on hERG endocytosis, we overexpressed
Cav-3 in human embryonic kidney (HEK) 293 cells stably expressing hERG
channels. Cav-3 overexpression decreased both the hERG current and the chan-
nel expression levels by more than 50% in normal culture conditions. In addi-
tion, Cav-3 overexpression exacerbated hERG endocytosis under hypokalemic
(0 mM Kþo) culture conditions. Since hypokalemia has also been shown to re-
duce hERG conductance in cardiomyocytes, we demonstrated that knockdown
of Cav-3 using siRNA can significantly impede such reduction. Co-immuno-
precipitation analysis and confocal imaging also demonstrated an association
between hERG and Cav-3 in rat and rabbit cardiomyocytes. Our data indicate
that Cav-3 participates in hERG trafficking, and is an important regulator of
hERG expression in cardiac myocytes. (Supported by the Heart and Stroke
Foundation of Ontario and Canadian Institutes of Health Research)
554-Pos Board B354
Cardiac PotassiumChannel hERG IsRegulated byUbiquitinLigase Nedd4l
Heidi Shallow, Jun Guo, Shetuan Zhang.
The cardiac rapidly activating delayed rectifier potassium channel (IKr) is en-
coded by the human ether-a-go-go related gene (hERG), which is important
for repolarization of the cardiac action potential. Reduction in hERG expres-
sion levels due to genetic mutations or drugs causes Long QT Syndrome
(LQTS). Recently, we demonstrated that ubiquitination of hERG channels is
involved in low Kþ induced hERG endocytic degradation. Since homeostatic
degradation is an important pathway in maintaining hERG membrane expres-
sion levels, we investigated the molecular mechanisms for hERG degradation
by focusing on the role and consequence of the ubiquitin (UB) ligase, Nedd4L.
Ub plays a role in the internalization of cell-surface hERG channels, and we
hypothesized that ubiquitination of hERG channels is facilitated through
Nedd4L. To study the effects of Nedd4L on hERG channels, we overexpressed
Nedd4L in human embryonic kidney (HEK) 293 cells that stably express the
hERG channels. We performed electrophysiological recordings, Western
blot, co-immunoprecipitation analysis, and confocal microscopy to identify
Nedd4L’s role in hERG expression. Our data from whole-cell patch clamp re-
cordings demonstrated that, among hEAG, Kv1.5 and hERG, Nedd4L specifi-
cally eliminates the hERG channel current. Western blot and confocal imaging
analyses showed that Nedd4L overexpression led to significant reduction in
mature hERG channels in the plasma membrane. Data obtained using co-im-
munoprecipitation indicated that Nedd4L significantly increases ubiquitinated
hERG channels. Our data indicate that Nedd4L may play a role in hERG ho-
meostatic degradation. (Supported by the Canadian Institutes of Health Re-
search and Heart and Stroke Foundation of Ontario).
555-Pos Board B355
Structural Basis of Action for a Novel hERG1 Channel Activator
Vivek Garg, Frank Sachse, Michael Sanguinetti.
The rapid delayed rectifier Kþ current (IKr) is conducted by human ether-a-
go-go-related gene 1 (hERG1) channels and plays a predominant role in the
repolarization of cardiac action potentials. Long QT syndrome caused by
loss-of-function mutations in hERG1, or unintended blockade of hERG1 chan-
nels by commonly used drugs, can lead to severe arrhythmia and sudden death.
Here, we investigated the structural basis of action of ICA-105574 (ICA),
a novel hERG1 agonist previously reported to activate current by causing
a large shift (>180 mV) in the V0.5 for P-type inactivation. Here we show
that ICA is a partial agonist and has decreased activity in inactivation-impaired
mutants of hERG1. Alanine-scanning mutagenesis of the S5, pore-helix and S6
segments of hERG1 and voltage clamp analysis of mutant channels expressed
in Xenopus oocytes was used to define residues critical for current activation by
ICA. The effects of 30 mM ICA were tested on 43 different mutant channels.
Nine mutations attenuated ICA-mediated hERG1 activation by >10-fold.
Two mutations, F557L (S5) and L622 (pore helix) completely prevented
hERG1 activation by ICA. Residues identified as critical for ICA activity


Activation and Complex Regulation of the Kef Potassium Efflux System During Protection of Bacteria Against Toxic Electrophiles 

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Activation and Complex Regulation of the Kef Potassium Efflux System During Protection of Bacteria Against Toxic Electrophiles

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