DEVELOPMENTAL REGULATION OF THE A-TYPE POTASSIUM-
CHANNEL CURRENT IN HIPPOCAMPAL NEURONS: ROLE OF THE
Kv1.1 SUBUNIT
T. FALK,
a
R. K. KILANI,
b
L. A. STRAZDAS,
b
R. S. BORDERS,
b
J. V. STEIDL,
a1
A. J. YOOL
a
AND
S. J. SHERMAN
a,b
*
a
Department of Physiology
b
Department of Neurology, The University of Arizona, 1501 North
Campbell Avenue, Tucson, AZ 85724, USA
Abstract—The rapidly inactivating A-type K
current (I
A
) is
prominent in hippocampal neurons; and the speed of its
inactivation may regulate electrical excitability. The auxiliary
K
channel subunit Kv1.1 confers fast inactivation to Shak-
er-related channels and is postulated to affect I
A
. Whole-cell
patch clamp recordings of rat hippocampal pyramidal neu-
rons in primary culture showed a developmental decrease in
the time constant of inactivation (
in
) of voltage-gated K
currents: 17.91.5 ms in young neurons (5–7 days in vitro;
n53, meanS.E.M.); 9.91.0 ms in mature neurons (12–15
days in vitro; n72, meanS.E.M., P<0.01). During the same
developmental time, the level of Kv1.1 transcript increased
more than two-fold in vitro and in vivo, determined by semi-
quantitative reverse transcriptase-polymerase chain reaction
for hippocampus. The hypothesis that up-regulation of
Kv1.1 led to the changes in
in
was tested in vitro, using
antisense knockdown. Kv1.1-specific antisense DNA was
introduced with a modified herpes virus co-expressing en-
hanced green fluorescent protein and knockdown of Kv1.1
was verified by immunocytochemistry. Following transduc-
tion with the antisense virus, mature neurons reverted to
in
values characteristic of young neurons: 18.32.4 ms (n20).
The effect of antisense knockdown on electrical excitability
was tested using current-clamp protocols to induce repetitive
firing. Treatment with the antisense virus increased the inter-
spike interval over a range of membrane depolarization
(baseline membrane potential40 to 20 mV). This effect
was most pronounced at 40 mV, where the ISI of the first
pair of action potentials was nearly doubled.
These data indicate that Kv1.1 contributes to the devel-
opmental control of I
A
in hippocampal neurons and that the
magnitude of effect is sufficient to regulate electrical excit-
ability. Viral-mediated antisense knockdown of Kv1.1 is ca-
pable of decreasing the electrical excitability of post-mitotic
hippocampal neurons, suggesting this approach has appli-
cability to gene therapy of neurological diseases associated
with hyperexcitability. © 2003 IBRO. Published by Elsevier
Science Ltd. All rights reserved.
Key words: antisense DNA, herpes simplex virus 1, voltage
clamp, gene therapy.
Profound changes in neuronal excitability occur within the
hippocampus during early postnatal brain development
(Johnston, 1996; Swann et al., 1990) and result from the
regulation of numerous individual genes. Defining the mo-
lecular basis of these changes is a formidable challenge,
but will be rewarding in our understanding of both normal
development and the pathophysiology of disorders such as
epilepsy. Voltage-gated K
+
channels play a fundamental
role in controlling neuronal excitability and are critical de-
terminants of the interspike interval (ISI) during repetitive
firing (Hille, 1992). In this regard, the degree and speed of
K
+
channel inactivation following an action potential is a
major regulatory influence in determining the firing rate.
The A-type current (I
A
), initially described in molluscan
systems, is a transient outward K
+
current that contributes
to mechanisms of learning and memory (Storm, 1987;
Johnston et al., 2000; Giese et al., 2001) and is prominent
in mammalian hippocampus (Hoffman et al., 1997). The I
A
current of the hippocampus is defined by its electrophysi-
ological characteristics of rapid inactivation following de-
polarization. Due to the immense diversity of K
+
channels,
it is likely that there are several subtypes of K
+
channels
that, in sum, give rise to the macroscopic I
A
current (Song,
2002). At the present time, there is a paucity of information
concerning which K
+
channels species contribute to I
A
in
different brain regions or in different stages of develop-
ment.
In this report, we studied the developmental time
course of Kv1.1, an auxiliary subunit of voltage-gated K
+
channels, and then correlated the level of expression of
this gene to the electrophysiological properties of develop-
ing hippocampal neurons. The K
+
channel complex con-
sists of tetrameric pore-forming -subunits and auxiliary
-subunits that play a modulatory role (Pongs et al., 1999).
A simplified nomenclature has been suggested which di-
vides the -subunits into 3 sub-families (Kv1.n, Kv2.1,
Kv3.1), Kv1.n having three members (England et al.,
1995). Kv1.n, and Kv3.1 subunits are known to regulate
inactivation properties of the channel complex (Rettig et
al., 1994) and Kv2.1 acts as a chaperone molecule
(Pongs et al., 1999) that guides sub-cellular localization of
-subunits. Since Kv1.1 associates with -subunits in the
1
Present address: Department of Drug Safety Evaluation, Pfizer, Inc.,
San Diego, CA, USA.
*Correspondence to: S. J. Sherman, Department of Neurology, The
University of Arizona, 1501 North Campbell Avenue, Tucson, AZ
85724, USA. Tel: +1-520-626-2319; fax: +1-520-626-5999.
E-mail address: ssherman@u.arizona.edu (S. J. Sherman).
Abbreviations: EGTA, ethylene glycol-bis (b-aminoethyl ether)-
N,N,N',N'-tetraacetic acid; eGFP, enhanced green fluorescent protein;
HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; I
A
, A-
type K
+
current; ISI, interspike interval; I
SS
, steady-state current; MEM,
minimal essential medium; PBS, phosphate-buffered saline;
in
, time
constant of inactivation; RT-PCR, reverse transcriptase polymerase
chain reaction; Vm, baseline membrane potential.
Neuroscience 120 (2003) 387– 404
0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0306-4522(03)00044-7
387