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Recent Work
(Link to
a complete list of
publications)
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Rescue of genetic mutant channels by
chemical activators
Xiong, Q., Sun, H., Nan,
F. and Li, M., Combinatorial Activation of KCNQ potassium channels by
synthetic chemical ligands. .
Proc. Natl. Acad. Sci.
USA 105,
3128-3133 (2008). (Full text
in PDF)
 Neuronal
M-currents play a critical role in controlling membrane excitability. They
are mediated by heteromultimeric channels comprised of KCNQ2 (Kv7.2) and
KCNQ3 (Kv7.3) subunits. Some mutations in these genes are causal to human
diseases, including benign familial neonatal convulsion (BFNC). Compounds
capable of potentiating M current/KCNQ channels would therefore be of great
interest. Retigabine (RTG) and zinc pyrithione (ZnPy) are two activators
for KCNQ channels with different pharmacological properties. However, their
molecular interactions with KCNQ channel remain largely elusive. It is not
known whether KCNQ channels may be modulated simultaneously by more than one
activator. Here we report that retigabine and zinc pyrithione recognize two
different sites of KCNQ2 channels. Their non-competitive agonistic
potentiation allows for simultaneous binding of two different activators on
the same channel complex, hence giving rise to combinatorial potentiation.
The effects of these activators are potent and diverse, capable of
activating a non-conductive channel and restoring properties of a human
mutant channel to characteristics resembling those of wild type.
Independent ligand binding sites and combinatorial potentiation by multiple
synthetic chemical openers underscore a wide range of regulation that could
be conferred to KCNQ potassium channels.
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Chemical biology approaches to
investigate neuronal ion channel function and therapeutic potential.
Qiaojie Xiong, Haiyan Sun
and Min Li. Novel ligand-mediated activation of voltage-gated KCNQ potassium
channels rescues epileptogenic mutants. Nature Chemical Biology
3, 287-296
(2007) (full text
download)
 KCNQ
potassium channels are important regulatory proteins
for controlling electrical excitability. Mutations of
KCNQ2 and KCNQ3 potassium channel genes result in reduction or loss of
potassium channel activity and cause benign familial neonatal
convulsions (BFNCs). Here we report a novel agonistic compound, zinc
pyrithione, which activates both recombinant and
native KCNQ M currents, that normally are opened by changes in transmembrane
voltage. At saturation voltage, increase of single
channel open probability (Po) could fully account for the
overall Gmax increase. Separate point mutations have been
identified either shifting the concentration dependence
or affecting potentiation efficacy, providing evidence supportive for key
residues influencing ligand binding and downstream
events. Furthermore, zinc pyrithione is capable of rescuing the mutant
channels causal to BFNCs. The ability of a new agonist to enhance the
potassium current through native M channels and augment the genetically defective KCNQ channels promises the potential
utilities in probing channel functions and developing therapeutics.
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Plasticity of forward trafficking signals - genetic
isolation and mechanistic characterization
Sojin Shikano, Brian
Coblitz, Haiyan Sun, and Min Li. Genetic selection of trafficking
signals that direct cell surface expression. Nature Cell Biol.
7, 985-992
(2005) (full text
download)
Membrane proteins represent
approximately 30% of the proteome in both prokaryotes and eukaryotes. The
spatial localization of membrane-bound proteins is often determined by
specific sequence motifs, that may be regulated in response to physiological
changes, such as protein interactions and receptor signaling. Identification
of signaling motifs is therefore important for understanding membrane
protein expression, function and transport mechanisms. We report a genetic
isolation of novel motifs that confer surface expression. Further
characterization showed that SWTY, one class of these isolated motifs with
homology to previously reported forward transport motifs, has the ability to
both override the RKR ER localization signal and potentiate steady-state
surface expression. The genetically isolated SWTY motif is functionally
interchangeable with a known motif in cardiac potassium channels and an
identified motif in an HIV co-receptor and operates by recruiting 14-3-3
proteins. This study expands the repertoire of and enables a screening
method for forward transport signals.
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 Acute
and chronic inhibition of cardiac
potassium channels - FRAC technologies and applications
Haiyan Sun, Xiaodong Liu,
Sojin Shikano,
and Min Li. Chronic inhibition of cardiac Kir2.1 and hERG potassium channels by
celastrol with dual effects on both ion conductivity and protein trafficking.
J. Biol. Chem.
281, 5877-5884 (2006) (full text
download)
A high percentage of
drugs and drug candidates has been found to cause cardiotoxicity by reducing
potassium conductance, more commonly known as QT prolongation. However, some
compounds do not show direct block of ionic flow, suggesting that other
mechanisms may also lead to reduction of potassium currents. Using the
functional recovery after chemobleaching (FRAC) assay, we have examined a
collection of drugs and drug-like compounds for potential perturbation of
cardiac potassium channel trafficking. Here we report that a significant number
of inhibitory compounds displayed effects on channel expression on the cell
surface. Further investigation of celastrol
(3-hydroxy-24-nor-2-oxo-1(10),3,5,7-friedelatetraen-29-oic acid), a
cell-permeable dienonephenolic triterpene compound, revealed its potent
inhibitory activity on both Kir2.1 and hERG potassium channels, causal to QT
prolongation. In addition to acute block of ion conduction, celastrol also
alters the rate of ion channel transport and causes a reduction of channel
density on the cell surface. In contrast, celastrol has no effects on
trafficking of either CD4 or CD8 membrane proteins. Furthermore, the potency
for reducing surface expression is approximately 5- to 10-fold more effective
than that for either direct acute inhibition or reported cytoprotectivity via
activation of the heat shock transcription factor 1. Because the reduction of
potassium channel activity is a common form of drug-induced cardiotoxicity, the
potent inhibition of cell surface expression by celastrol underscores a need to
evaluate drug candidates for their chronic effects on biogenesis of potassium
channels. Our results suggest that chronic exposure to certain drugs may be an
important aspect of acquired QT-prolongation.
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 Carboxyl-terminal
recognition by 14-3-3 proteins
Brian Coblitz, Sojin Shikano,
Meng Wu, Sandra Gabelli, Lisa Cockrell, Matt Spieker, Yoshiro Hanyu, Haian Fu,
L. Mario Amzel and Min Li. C-terminal recognition by 14-3-3 proteins for
surface expression of membrane proteins. J. Biol. Chem. 280,
36263-36272 (2005) (full text
download)
Diverse
functions of 14-3-3 proteins are directly coupled to their ability to interact
with targeted peptide substrates. RSX(pS/pT)XP and RXFX(pS/pT)XP
are two canonical consensus binding motifs for 14-3-3 proteins representing the
two common binding modes, modes I and II, between 14-3-3 and internal peptides.
Using a genetic selection, we have screened a random peptide library and
identified a group of C-terminal motifs, termed SWTY, capable of overriding an
ER localized signal and re-directing membrane proteins to cell surface. Here we
report that the carboxyl-terminal SWTY motif, although different from modes I
and II consensus, binds tightly to 14-3-3 proteins with a dissociation constant
(Kd) of 0.17 mM, comparable to that
of internal canonical binding peptides. We show that all residues but proline
in -SWTX-COOH are compatible for the interaction and surface expression.
Because SWTY-like sequences have been found in native proteins, these results
support a broad significance of 14-3-3 interaction with protein C-termini. The
C-terminal binding consensus, mode III, represents an expansion of the
repertoire of 14-3-3-targeted sequences.
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