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The Journal of Neuroscience, March 1, 1999, 19(5):1691-1697
Inducible Genetic Suppression of Neuronal Excitability
David C.
Johns1,
Ruth
Marx2,
Richard E.
Mains2,
Brian
O'Rourke1, and
Eduardo
Marbán1
1 Section of Molecular and Cellular Cardiology,
Division of Cardiology, and 2 Department of Neuroscience,
Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
Graded, reversible suppression of neuronal excitability represents
a logical goal of therapy for epilepsy and intractable pain. To achieve
such suppression, we have developed the means to transfer "electrical
silencing" genes into neurons with sensitive control of transgene
expression. An ecdysone-inducible promoter drives the expression of
inwardly rectifying potassium channels in polycistronic adenoviral
vectors. Infection of superior cervical ganglion neurons did not affect
normal electrical activity but suppressed excitability after the
induction of gene expression. These experiments demonstrate the
feasibility of controlled ion channel expression after somatic gene
transfer into neurons and serve as the prototype for a novel
generalizable approach to modulate excitability.
Key words:
genetic; neuronal excitability; viral gene transfer; inducible expression; gene therapy; adenovirus
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INTRODUCTION |
Excessive cellular excitability
underlies a variety of common, often lethal diseases, ranging from
epilepsy to cardiac ventricular arrhythmias. Conventional therapeutic
efforts to suppress excitability in such disorders center around the
pharmacological block of selected ion channels, mechanical disruption
of an irritable focus, or devices such as pacemakers or defibrillators.
The drug block of ion channels, although sometimes effective, is
plagued by side effects attributable to the systemic administration of
drugs needed only in small areas of the body (Jallon, 1997 ; Loscher,
1998). Mechanical disruption of epileptic or arrhythmic foci is
traumatic, irreversible, and often ineffective. Implantable devices
designed to terminate hyperexcitation are expensive and are activated
only after the seizure has begun. We have explored inducible genetic suppression as an alternative approach to the treatment and prevention of hyperexcitability. Overexpression of inwardly rectifying potassium channel genes driven by an inducible promoter inhibits evoked and
spontaneous activity in cultured neurons. Blocking the expressed channels reverses the suppression of excitability. The findings demonstrate that induction of exogenous hyperpolarizing genes effectively suppresses excitability. The inducible expression of such
genes represents a new therapeutic principle for use in disorders of
hyperexcitability and provides a powerful research tool for probing the
contributions of specific regions of the nervous system to the global
functional response.
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MATERIALS AND METHODS |
Plasmid vectors. The coding sequence for the human
Kir2.1 gene was amplified using the PCR to allow in-frame fusion
to enhanced green fluorescent protein (EGFP) in the vector pEGFP-C3
(Clontech, Palo Alto, CA) to create pEGFP-Kir2.1. Kir2.1 was also
cloned into the vector pGFP-IRES (Johns et al., 1997) to create
the vector pGFP-IRES-Kir2.1. The adenovirus shuttle vector pAdLox
(Hardy et al., 1997) was modified to replace the cytomegalovirus (CMV) promoter with the ecdysone-inducible promoter from pIND-1 (Invitrogen, San Diego, CA), making the vector pAdEcd. The expression cassettes from
pGFP-IRES, pGFP-IRES-Kir2.1, and pEGFP-Kir2.1 were cloned into the
multiple cloning site of pAdEcd, making the vectors pAdEGI, pAdEGI-Kir2.1, and pAdEG-Kir2.1. The plasmid pAdVgRXR was made by
cloning the expression cassettes from pVgRXR (Invitrogen, San Diego,
CA) into pAdLox, encoding the ecdysone receptor.
Cell culture. A549, 293, and CRE8 cells (CCL-185 and
CRL-1573; American Type Culture Collection, Rockville, MD; Hardy et al. (1997), respectively) were grown in DMEM (CellGro; Mediatech, Washington, DC) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamax, 1× penicillin-streptomycin, and 15 mM HEPES, pH 7.4 (Life Technologies, Gaithersburg, MD), at
37C° in a 5% CO2, humidified incubator.
Viral vectors. Recombinant adenovirus vectors were generated
by Cre-lox recombination (Hardy et al., 1997). A 25 cm2 flask (T25; Sarstedt, Newton, NC) of CRE8 cells
was cotransfected with 2.1 µg of purified  5 viral DNA and 2.1 µg
of purified shuttle vector DNA using Lipofectamine Plus (Life
Technologies). Cells were incubated 5-9 d until cytopathic effects
(CPE) were observed. Cells and media were transferred to a 50 ml
polypropylene tube (Sarstedt) and freeze-thawed in a dry ice-ethanol
bath and a shaking 37C° water bath. Cells and debris were removed by
centrifugation in a clinical centrifuge, and 2 ml of the supernatant
was added to a 90% confluent T25 and returned to the incubator until
CPEs were observed. This procedure was repeated three to four times at
which time the virus was analyzed for purity by plaque assays. Viruses
were then expanded, and large-scale virus purifications were performed
as described previously (Johns et al., 1995). Virus titers were
determined by plaque assays, and particle numbers were determined by
absorbance at 260 nm on a DU640 spectrophotometer (Beckman Instruments,
Fullerton, CA). As has been reported previously (Mittereder et al.,
1996), the particle number exceeded the infective particle number by
between 40- and 100-fold. Multiplicities of infection (mois)
were calculated on the basis of the number of plaque-forming units per milliliter.
Neuronal cultures. Superior cervical ganglion (SCG)
neurons were prepared by enzymatic dissociation from ganglia taken from newborn rat pups (Marek and Mains, 1989). Neurons were plated onto
glass coverslips coated with rat tail collagen (Bornstein, 1958).
Neurons were grown in DMEM/F12 containing 10% FBS and were treated
with 10 µM cytosine arabinoside starting 24 hr after
plating. Culture media contained  -nerve growth factor at 100 ng/ml
(Boehringer Mannheim, Indianapolis, IN, or kindly supplied by Dr. David
Ginty, Johns Hopkins University, Baltimore, MD).
Infections. Cells (A549 or primary SCG neurons) were
infected by replacing their normal growth media with DMEM with 2% FBS and the appropriate amount of virus for 2-4 hr. The amount of virus
used for A549 cells was varied as described for each experiment, whereas the amount of virus used for SCG neurons was held constant at
~50 moi of the test virus and 5 moi of the receptor virus. Infection
medium was then replaced with normal growth medium. Expression was
induced by addition of muristerone A [1 µM (see Fig. 1B) and 3 µM in all other
experiments; Invitrogen] for 22 hr (A549 cells) or 48 hr (SCG neurons)
before assays were performed. The dose of muristerone A was selected to
activate the receptor maximally on the basis of previously reported
dose-response curves (No et al., 1996), which agreed with our
own preliminary dose-response curves (data not shown).
Immunohistochemistry. Infected SCG neurons were fixed and
stained as described previously (Paquet et al., 1996; Marx and Mains, 1997; Milgram et al., 1997).
Confocal microscopy. Images were taken on a laser-scanning
confocal microscope (PCM 2000; Nikon, Melville, NY; excitation, 488 nm;
emission, 505-530 nm) with a 60× objective lens (numerical aperture, 1.2).
Electrophysiology. Experiments were performed at 21-23°C.
Whole-cell currents were recorded with an Axopatch 1-D amplifier (Axon
Instruments, Foster City, CA) sampled at 10 kHz and filtered at 1-5
kHz. The pipette solution contained (in mM): 140 K-glutamate, 1 MgCl2, 4 EGTA, 4 MgATP, and 10 HEPES,
pH 7.2. The bath solution for neurons contained (in mM):
140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4; for A549
cells, 140 KCl was substituted for NaCl.
Statistics. Pooled data are shown as means ± SEM. Data were analyzed using two-factor ANOVA. Between-group
comparisons were made post hoc with the Fisher's
least-significant-difference test.
Flow cytometry. Single-color flow cytometry was used to
determine the mean fluorescence intensity of infected A549 cells. Cells
were induced 1 d after infection; 22 hr later, cells were trypsinized, washed, and resuspended in 0.5 ml of PBS. Cells were analyzed on a FacStar (Becton Dickinson, Cockeysville, MD) using forward and side scatter gates to exclude dead cells and cell fragments
and collecting 1 × 104 events per sample.
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RESULTS |
Inducible viral constructs
To achieve sensitive control of gene expression after somatic gene
transfer, we created adenoviruses that enabled ecdysone-inducible expression of ion channels (No et al., 1996) and reporter genes in
mono- or polycistronic vectors (Fig.
1A). The polycistronic vector, as shown, results in an ~3:1 ratio of green fluorescent protein (GFP) to ion channel protein (Dirks et al., 1993),
allowing sensitive detection of cells without undue overexpression of
the ion channel. Infection of A549 cells with a stoichiometric ratio of
10 ecdysone-driven reporter viruses (AdEGI) per ecdysone/retinoid X
receptor virus (AdVgRXR) produced low basal levels of expression of GFP
but >15-fold enhancement after addition of the ecdysone analog
muristerone A (Fig. 1B). Excess levels of receptor
virus lead to higher levels of basal (noninduced) expression (Fig.
1B) as well as a slight decrease in the fold
induction achievable. This decrease in the level of induction may be in
part attributable to a higher percentage of induced cells above the
saturation level of the flow cytometer. The absolute level of
expression was increased with increasing levels of the receptor.
Removal and readdition of muristerone A (Fig. 1C) could turn
expression off and then on again, respectively. To test for
inducibility with channel-containing constructs, we coinfected A549
cells with an adenovirus that expresses both GFP and Kir2.1
(AdEGI-Kir2.1; moi = 5) and AdVgRXR (moi = 0.5). When
induced, 80-90% of the cells were GFP-positive in contrast to <1%
of infected but noninduced cells (based on flow cytometry measurements;
data not shown). Membrane current recordings from infected but
noninduced cells showed no inwardly rectifying current, whereas induced
cells expressing GFP exhibited robust inwardly rectifying currents
(Fig. 1D). Overexpression of such channels under
physiological conditions in neurons would be predicted to hyperpolarize
cells and to suppress excitability.
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Figure 1.
Ecdysone-inducible adenovirus constructs allow
tight control of gene expression. A, Schematic
representation of the basis for the ecdysone-inducible adenovirus
vectors is shown.  , Packaging signal; Ecd
promoter, ecdysone-inducible promoter;
IRES, internal ribosome entry site; ITR,
inverted terminal repeat; MCS, multiple cloning site;
pA, SV40 polyadenylation signal; RSV,
Rous sarcoma virus; RXR, retinoid X receptor;
VgEcR, modified ecdysone receptor.
B, Infection of A549 cells with variable mois of
both an adenovirus that contains an ecdysone-inducible promoter
controlling expression of a cassette containing GFP, an IRES,
and a multiple cloning site (AdEGI) and an adenovirus that expresses
the modified ecdysone receptor and the retinoid X receptor (AdVgRXR)
resulted in muristerone A-dependent and -inducible expression of GFP
( vs  ). In the absence of AdVgRXR, background expression was
minimal ( ), and such cells were entirely refractory to muristerone A
( ). C, Expression of GFP in A549 cells, infected with
both AdVgRXR and AdEGI, can be induced with 3 µM
muristerone A for the indicated amounts of time (columns
2-4), reduced to near background levels by the removal
of muristerone A for 36 hr (column 5), and reinduced to
the original induction level with muristerone A for 24 hr
(column 6). D, Membrane currents
from A549 cells infected with AdVgRXR and AdEGI-Kir2.1 in the
absence (I =  5 pA/pF;
n = 2) and presence (I = 80 ± 12 pA/pF; n = 4) of 3 µM
muristerone A are shown.
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Infection of neuronal cells
To confirm efficient infection and inducibility in excitable
cells, we used neurons cultured from neonatal rat SCGs. SCG
neurons have been used extensively for studies of electrophysiology,
biochemistry, and development (Argiro and Johnson, 1982; Nerbonne and
Gurney, 1989; Garyantes and Regehr, 1992; McFarlane and Cooper, 1992; Lockhart et al., 1997); in addition, these neurons are known to be
amenable to adenoviral gene transfer (Paquet et al., 1996). SCG neurons
infected with AdEGI-Kir2.1 and AdVgRXR for 48 hr and treated with
vehicle for 22 hr exhibited negligible GFP fluorescence (Fig.
2A), in contrast with
cells imaged 22 hr after the addition of muristerone A (Fig.
2B). A vector containing a GFP-Kir2.1 fusion protein (AdEG-Kir2.1) enabled us to track the distribution of the
expressed channel complex. Comparison of the distribution of GFP (Fig.
2D) with that of microtubule-associated protein 2 (MAP2; Fig. 2C) showed substantial overlap (Fig.
2E); indeed, Kir2.1 protein was distributed
throughout the surface and internal membranes of the neurons. In these
experiments, 52% of the MAP2-positive cells were also GFP positive
(n = 100); in addition, all of the glial cells that
could be identified by visible light were GFP positive.
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Figure 2.
Infection of SCG neurons with inducible adenovirus
constructs. A, B, Confocal images of SCG
neurons infected with AdEGI-Kir2.1 and AdVgRXR in the absence
(A) or presence (B) of
muristerone A. C-E, A neuron expressing the fusion
protein EGFP-Kir2.1 after fixation and staining with a polyclonal
antibody to GFP and a monoclonal antibody against the MAP2. An
FITC-labeled goat anti-rabbit secondary antibody was used to detect the
presence of GFP (D), and a Cy3-conjugated
secondary antibody was used to detect the bound anti-MAP2
(C). MAP2 is normally distributed in the
dendrites and cell bodies.
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Suppression of neuronal excitability
In the process of characterizing the phenotypic consequences of
Kir2.1 gene transfer and induction, we first compared action potentials
and membrane currents in AdEGI-infected and uninfected SCG neurons and
found no differences; data from these two groups were thus combined as
"control cells" (data not shown). Barium (Ba2+)
selectively blocks inwardly rectifying potassium channels, which are
relatively sparse in control SCG neurons (Wang and McKinnon, 1995).
Stimulated action potentials in a control cell were identical in the
absence or presence of Ba2+ (Fig.
3A,B),
as were membrane currents (Fig. 3C). In contrast, neurons
expressing Kir2.1 exhibited diminished excitability. Inward current
injection produced only a subthreshold depolarization in an induced
cell infected with AdEGI-Kir2.1 (Fig. 3D); after the
addition of 50 µM Ba2+, the resting
potential depolarized, and a current stimulus of the same intensity
evoked a full action potential (Fig. 3E). The current-voltage relations in this cell reveal marked inward
rectification at baseline; the addition of Ba2+ made
the membrane currents indistinguishable from those in control cells
(Fig. 3F). In addition to reshaping evoked activity,
induced Kir2.1 gene expression also suppressed spontaneous electrical activity (Fig. 3G-I). Six of 14 control cells
exhibited spontaneous activity in normal solution, but none of the 14 induced Kir2.1-infected cells did so. Nevertheless, 5 of these 14 neurons began firing spontaneously after superfusion with 50 µM Ba2+. The cell shown in Figure 3,
G-I, expressed only 100 pA of
Ba2+-sensitive current at 120 mV, revealing that
the amount of expression needed to achieve biologically relevant
suppression was small. On the other hand, cells with as much as 1.1 nA
of current also exhibited Ba2+-induced spontaneous
activity, indicating that high-level expression did not irreversibly
inhibit the ability of the neuron to respond to normal synaptic
input.
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Figure 3.
Exogenous expression of Kir2.1
suppresses excitability. A, B, The
effects of a suprathreshold stimulus on a control cell do not change
with the addition of barium. C, The membrane current
recorded from the same cell under these conditions using a ramp
protocol from 138 to +22 mV over 500 msec is shown. The addition of
barium had little or no detectable effect on the outwardly rectifying
I-V curves in control cells. D,
E, The response to a subthreshold stimulus on a
Kir2.1-infected cell (D) is markedly changed by
the addition of barium (E); the
arrow indicates the cessation of the applied stimulus.
F, The I-V curve for this cell is much
different after addition of barium. G-I,
Membrane current recordings from a Kir2.1-infected cell are shown in
normal saline (G), after superfusion with saline
containing barium (H), and after washout
(I).
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Figure 4 summarizes the changes in
resting membrane potential (A) and stimulus threshold
(B) effected by induction of Kir2.1 expression. In
the absence of Ba2+, control cells were depolarized
relative to Kir2.1-induced cells (63 vs 85 mV; p < 0.0001). Some (but not all) of the control cells exhibited a small
depolarization in response to Ba2+, presumably
because of the block of low-density native inward rectifiers. In
contrast, every Kir2.1-induced cell depolarized after exposure to
Ba2+ (p < 0.001). Figure
4B quantifies the change in excitability as the
difference between the threshold amount of stimulus current needed to
fire an action potential in the absence or presence of 50 µM Ba2+. The stimulus threshold was
Ba2+ insensitive in control cells. In general,
Kir2.1-induced cells required dramatically greater stimuli to reach
threshold in the absence of Ba2+
(p < 0.0005). To examine the effects of the
varying densities of expressed Kir2.1, we divided the cells into two
groups based on the amount of Ba2+-sensitive current
they exhibited (600 pA was the midpoint; range, 100-1100 pA). The
increase in stimulus threshold was distinctly greater in the cells that
expressed a higher density of Kir2.1.
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Figure 4.
Cumulative effects of exogenous expression of
Kir2.1. A, Cells expressing Kir2.1 are hyperpolarized
with respect to control cells. This effect is reversed after blocking
the Kir2.1 channels. B, Increasing amounts of
Kir2.1 current increase the size of the stimulus needed to fire an
action potential.
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DISCUSSION |
Our results demonstrate the ability to modify neuronal
excitability genetically in an inducible and reversible manner. The ecdysone regulatory system has little background activity when appropriate amounts of receptor virus are used and yet is inducible to
high levels. Under the conditions described, there was an inherently high level of variability of expression among infected cells. This was
the result of coinfecting with less than saturating amounts of both the
therapeutic virus and the receptor virus. Figure 1B shows that increasing the number of the receptor viruses per cell, while holding the number of test viruses constant, increases the amount
of expression. Similar experiments varying the number of test viruses
delivered, while holding the number of receptor virus constant, showed
that this also affected expression levels (data not shown). The
variable levels of GFP fluorescence visible in infected cells confirmed
these predictions. For these reasons we did not perform dose-response
experiments in neurons, in which the level of muristerone A was also
varied. One potential refinement would be to include the receptor
complex on the same vector, perhaps under the control of a
tissue-specific promoter, to accomplish more uniform tissue-specific
expression. The use of the IRES was quite beneficial in allowing easy
detection of positive cells without having to increase the amount of
ion channel expression to levels that are not physiologically relevant.
These properties enabled us to examine a wide range of
electrophysiological effects. On one end, low levels of expression of
Kir2.1 sufficed to inhibit spontaneous activity (Fig.
3G-I) despite a relatively modest 66% increase in
the current needed to reach threshold. At the other extreme, two cells,
which were not included in the analysis, exhibited Ba2+-sensitive currents >1.5 nA. In these two
cells, action potentials could not be elicited when a maximal current
stimulus (2 nA) was applied for up to 50 msec; nevertheless, in the
presence of Ba2+, both of these neurons fired action
potentials with modest stimuli. Such stimulus currents far exceed the
probable summed inputs for any single neuron and thus represent a
complete silencing of electrical activity.
The higher infection efficiency of the glial cell population (100%)
versus the neuronal population (52%) has been observed previously by
us as well as others (Moriyoshi et al., 1996; Johns et al.,
1997). This represents another potential limitation of this system, in
that there may indeed be profound effects of hyperpolarizing the
supporting cell population. To avoid this complication in vivo, several strategies could be used. These include the
previously mentioned use of neuronal-specific promoters, of vectors
modified to increase neuronal tropism (Douglas et al., 1996; Rogers et al., 1997; Dmitriev et al., 1998), or of vectors that are naturally more neurotropic (Douglas et al., 1996; Rogers et al., 1997; Dmitriev et al., 1998).
The potential applications for this technology are widespread. The role
of ion channels in nervous system development has been explored
previously by the use of blocking reagents (Kater and Mills, 1991;
Rakic and Komuro, 1995). Using this strategy may enable genetic
manipulation of excitability at different points in development and
potentially in specific regions of the CNS. From a clinical standpoint,
this targeted and controllable delivery of ion channels may form the
basis for new treatments of diseases of excitability such as epilepsy
(Wasterlain et al., 1996). In addition, targeted delivery of these (or
similar) vectors (Liu et al., 1997) would result in controllable
suppression of excitability with the potential for genetic analgesia in
the treatment of intractable pain. The ability to turn gene expression
on and off at will enables the titration of responses while preserving
remarkable safety; if therapy should prove detrimental or ineffective,
the inducing agent could simply be withdrawn.
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FOOTNOTES |
Received Sept. 18, 1998; revised Nov. 19, 1998; accepted Dec. 9, 1998.
Dedicated to the memory of Sir Alan Hodgkin.
This study was supported by Tanabe Seiyaku Company and by National
Institutes of Health Grant DA-00266. We thank Dr. S. Hardy for the
Cre-lox adenoviral vectors and for helpful discussions.
Correspondence should be addressed to Dr. Eduardo Marbán, Section of
Molecular and Cellular Cardiology, 844 Ross Building, The Johns Hopkins
University School of Medicine, 720 North Rutland Avenue, Baltimore, MD 21205.
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D. L. Chao and G. J. Wang
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H. Luan, W. C. Lemon, N. C. Peabody, J. B. Pohl, P. K. Zelensky, D. Wang, M. N. Nitabach, T. C. Holmes, and B. H. White
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Hypothalamic Growth Hormone-Releasing Hormone (GHRH) Deficiency: Targeted Ablation of GHRH Neurons in Mice Using a Viral Ion Channel Transgene
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S. Naik, C. K. Billington, R. M. Pascual, D. A. Deshpande, F. P. Stefano, T. A. Kohout, D. M. Eckman, J. L. Benovic, and R. B. Penn
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M. Murata, E. Cingolani, A. D. McDonald, J. K. Donahue, and E. Marban
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E. S. Mohammadi, E. A. Ketner, D. C. Johns, and G. Ketner
Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line
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H. S. Yoon and V. W. Yang
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P. S. Lange, F. Er, N. Gassanov, and U. C. Hoppe
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B. A. Alseikhan, C. D. DeMaria, H. M. Colecraft, and D. T. Yue
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E. M. Slimko, S. McKinney, D. J. Anderson, N. Davidson, and H. A. Lester
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W. Guo, S. A. Malin, D. C. Johns, A. Jeromin, and J. M. Nerbonne
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H. Nadeau and H. A. Lester
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H. A. E. Lechner, E. S. Lein, and E. M. Callaway
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S. Giovannardi, G. Forlani, M. Balestrini, E. Bossi, R. Tonini, E. Sturani, A. Peres, and R. Zippel
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C. J. McCormick, D. J. Rowlands, and M. Harris
Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors
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Q. Cheng, P. M. Burkat, J. C. Kulli, and J. Yang
GABACrho 1 Subunits Form Functional Receptors But Not Functional Synapses in Hippocampal Neurons
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C. B. Chan, D. De Leo, J. W. Joseph, T. S. McQuaid, X. F. Ha, F. Xu, R. G. Tsushima, P. S. Pennefather, A. M. F. Salapatek, and M. B. Wheeler
Increased Uncoupling Protein-2 Levels in {beta}-cells Are Associated With Impaired Glucose-Stimulated Insulin Secretion: Mechanism of Action
Diabetes,
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[Abstract]
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Q. Cheng, J. C. Kulli, and J. Yang
Suppression of Neuronal Hyperexcitability and Associated Delayed Neuronal Death by Adenoviral Expression of GABAC Receptors
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R. A Li, J. Miake, U. C Hoppe, D. C Johns, E. Marban, and H B. Nuss
Functional consequences of the arrhythmogenic G306R KvLQT1 K+ channel mutant probed by viral gene transfer in cardiomyocytes
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U. C. Hoppe, E. Marban, and D. C. Johns
Distinct gene-specific mechanisms of arrhythmia revealed by cardiac gene transfer of two long QT disease genes, HERG and KCNE1
PNAS,
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[Abstract]
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R. A. Baines, J. P. Uhler, A. Thompson, S. T. Sweeney, and M. Bate
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R. El Meskini, L. Jin, R. Marx, A. Bruzzaniti, J. Lee, R. B. Emeson, and R. E. Mains
A Signal Sequence Is Sufficient for Green Fluorescent Protein to Be Routed to Regulated Secretory Granules
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C.-C. Shieh, M. Coghlan, J. P. Sullivan, and M. Gopalakrishnan
Potassium Channels: Molecular Defects, Diseases, and Therapeutic Opportunities
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H. Nadeau, S. McKinney, D. J. Anderson, and H. A. Lester
ROMK1 (Kir1.1) Causes Apoptosis and Chronic Silencing of Hippocampal Neurons
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M. A. Otieno and T. W. Kensler
A Role for Protein Kinase C-{{delta}} in the Regulation of Ornithine Decarboxylase Expression by Oxidative Stress
Cancer Res.,
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M. T. Perez-Garcia, J. R. Lopez-Lopez, A. M. Riesco, U. C. Hoppe, E. Marban, C. Gonzalez, and D. C. Johns
Viral Gene Transfer of Dominant-Negative Kv4 Construct Suppresses an O2-Sensitive K+ Current in Chemoreceptor Cells
J. Neurosci.,
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M. Sasaki, M. Gonzalez-Zulueta, H. Huang, W. J. Herring, S. Ahn, D. D. Ginty, V. L. Dawson, and T. M. Dawson
Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism
PNAS,
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J. Seharaseyon, N. Sasaki, A. Ohler, T. Sato, H. Fraser, D. C. Johns, B. O'Rourke, and E. Marban
Evidence against Functional Heteromultimerization of the KATP Channel Subunits Kir6.1 and Kir6.2
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E. Marban
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Circulation,
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Z.-Q. Xiong and J. L. Stringer
Cesium Induces Spontaneous Epileptiform Activity Without Changing Extracellular Potassium Regulation in Rat Hippocampus
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R. Marx, R. El Meskini, D. C. Johns, and R. E. Mains
Differences in the Ways Sympathetic Neurons and Endocrine Cells Process, Store, and Secrete Exogenous Neuropeptides and Peptide-Processing Enzymes
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H. Hu, T. Sato, J. Seharaseyon, Y. Liu, D. C. Johns, B. O'Rourke, and E. Marbán
Pharmacological and Histochemical Distinctions Between Molecularly Defined Sarcolemmal KATP Channels and Native Cardiac Mitochondrial KATP Channels
Mol. Pharmacol.,
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X. Chen, D. C. Johns, D. E. Geiman, E. Marban, D. T. Dang, G. Hamlin, R. Sun, and V. W. Yang
Kruppel-like Factor 4 (Gut-enriched Kruppel-like Factor) Inhibits Cell Proliferation by Blocking G1/S Progression of the Cell Cycle
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W. Lee-Kwon, D. C. Johns, B. Cha, M. Cavet, J. Park, P. Tsichlis, and M. Donowitz
Constitutively Active Phosphatidylinositol 3-Kinase and AKT Are Sufficient to Stimulate the Epithelial Na+/H+ Exchanger 3
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E. Saez, M. C. Nelson, B. Eshelman, E. Banayo, A. Koder, G. J. Cho, and R. M. Evans
Identification of ligands and coligands for the ecdysone-regulated gene switch
PNAS,
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Top
Abstract
Introduction
