From the Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254
The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment
with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to
phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3
when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch
recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with
an antibody to the EGFr ligand binding domain (-EGFr), which blocks receptor dimerization and tyrosine kinase
activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type
inactivation, while pretreatment with the
-EGFr slows C-type inactivation. In contrast, insulin does not influence
C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation
rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.
Receptor tyrosine kinases play essential roles in cell
growth and differentiation, mitogenesis, and cell signaling (Schlessinger and Ullrich, 1992). These membrane receptors dimerize and/or alter their configuration upon ligand binding, resulting in kinase activation and the autophosphorylation of several COOH-terminal tyrosine residues. The phosphorylation of these residues facilitates the recruitment of specific proteins
containing Src homology 2 domains, which in turn initiates downstream signaling cascades that affect a diverse group of cellular targets via both tyrosine and
serine/threonine protein kinases. In particular, activation of the epidermal growth factor receptor (EGFr)1
and the insulin receptor tyrosine kinases is known to induce activation of PLC
, PLA2, PI-3 kinase and the ras/
MAPK (mitogen-activated protein kinase) pathway (Clark
and Dunlop, 1991
; Schlessinger and Ullrich, 1992
; Dikic
et al., 1994
). Tyrosine kinases, including the EGF and
insulin receptors, are expressed at high levels in the
nervous system, where they exhibit distinct and highly
regionalized patterns of distribution both during development and in the adult (Hanley, 1988
). In spite of
this, little is known about the signaling functions of tyrosine kinases in neurons, although emerging evidence from several laboratories indicates that ion channels
are among the targets of tyrosine kinases (reviewed by
Siegelbaum, 1994
; Jonas and Kaczmarek, 1996
). For example, in spinal dorsal horn neurons, the activity of
NMDA (N -methyl-D-aspartate) glutamate receptors is
increased by tyrosine kinases and decreased by tyrosine phosphatases (Wang and Salter, 1994
). In Aplysia bag
cell neurons, a voltage-dependent cation channel is
regulated by tyrosine phosphorylation, via a pathway involving serine/threonine kinases and phosphatases (Wilson and Kaczmarek, 1993
), while a calcium and potassium channel, as well as neuropeptide secretion, are
modulated by insulin (Jonas et al., 1996
, 1997
).
Voltage-dependent potassium (Kv) channels are important for the generation and regulation of electrical
activity in neurons, as well as for maintaining the resting membrane potential in excitable and nonexcitable
cells (reviewed by Hille, 1992). Based on sequence homology, Kv channels can be divided into several subfamilies; the Kv1 subfamily exhibits sequence homology
to the Drosophila Shaker potassium channel (Chandy,
1991
; Chandy and Gutman, 1993
). One important feature that distinguishes different Kv channels is their inactivation kinetics. Inactivation of Kv channels occurs by two distinct mechanisms. Fast or N-type inactivation
generally occurs with a time constant of a few milliseconds via an open channel pore block by the NH2-terminal region of the channel protein (Hoshi et al., 1990
).
C-type inactivation usually occurs with a time constant
of hundreds or thousands of milliseconds (Choi et al.,
1991
), by a less well defined mechanism that probably involves a more global change in the channel protein
conformation. Most Kv channels undergo C-type inactivation (although the time constant of inactivation can
vary widely (Lopez-Barneo et al., 1993
), while N-type inactivation is limited to those channels that possess the
appropriate NH2-terminal sequence or are associated with a
subunit containing a similar sequence (Rettig
et al., 1994
).
Studies during the last decade have made it evident
that Kv channels are important targets for modulation
by protein phosphorylation. Most work to date has focused on modulation by serine/threonine phosphorylation (reviewed in Levitan, 1994), but several Kv1 family members are also affected directly by tyrosine phosphorylation. In particular, Kv1.2 is suppressed by the
tyrosine kinase PYK2 and EGFr (Huang et al., 1993
; Lev
et al., 1995
; Peralta, 1995
) and Kv1.5 is suppressed by
Src (Holmes et al., 1996b
), as well as by activation of several growth factor receptors (Timpe and Fantl, 1994
).
Kv1.3 is phosphorylated directly by nonreceptor tyrosine kinases, resulting in suppression of channel activity and modulation of channel kinetics (Holmes et
al., 1996a
; Fadool et al., 1997
). Serine/threonine kinases
can also be involved in the modulatory pathway leading
to Kv channel modulation (Timpe and Fantl, 1994
).
In situ hybridization experiments indicate that Kv1
family members are expressed widely in mammalian
brain. One of these channels, Kv1.3, exhibits a particularly selective distribution in the olfactory bulb and olfactory cortex as well as in the dentate gyrus of the hippocampus (Kues and Wunder, 1992). Kv1.3 also is the
major Kv channel in T lymphocytes, where it plays an
essential role in cell proliferation and activation by antigens (Lewis and Cahalan, 1990
, 1995
). We have examined the modulation of Kv1.3 channels by the EGFr
and insulin receptor tyrosine kinases. Activation of either receptor tyrosine kinase decreases the peak Kv1.3
current amplitude. In addition, EGF treatment produces a speeding of C-type inactivation kinetics, while
insulin treatment does not alter C-type inactivation.
Mutational analysis indicates that different molecular
mechanisms mediate the effects of EGF on current amplitude and kinetics. Thus Kv channels are targets for
complex modulation by growth factor receptor tyrosine
kinases.
Materials
Human recombinant EGF and erbstatin were purchased from Calbiochem Inc. (La Jolla, CA), while insulin and a neutralizing EGFr antibody were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Cell culture reagents were from Gibco Laboratories (Grand Island, NY). Other chemicals were from Sigma Chemical Co. (St. Louis, MO).
Channel and Kinase Expression
The voltage-gated potassium channel Kv1.3 and the human EGFr were expressed transiently in HEK 293 cells. HEK 293 cells were maintained in MEM (12360; Gibco Laboratories) plus 2% penicillin/streptomycin (15140; Gibco Laboratories), and 10% fetal bovine serum (16000; Gibco Laboratories). Cells were grown to confluency (7 d), dissociated with trypsin-EDTA (Gibco Laboratories) and mechanical trituration, diluted in MEM to a concentration of ~600 cells/µl, and replated on plastic dishes. Cells were allowed to recover for several days before transfection. Kv1.3 was expressed using the Invitrogen Corp. (San Diego, CA) vector pRC-CMV, and the EGFr was expressed in the GW1 vector (British Biotechnology Ltd., Oxford, UK). In both cases, the protein coding region was downstream from a cytomegalovirus promoter. The effect of stimulating endogenous insulin receptors was studied by singly transfecting HEK 293 cells with Kv1.3 and adding insulin to the medium.
cDNA was introduced into cells with a lipofectamine reagent
(18324; Gibco Laboratories), using standard techniques (Hawley-Nelson et al., 1993). The lipofectamine and the cDNA(s) were
mixed and allowed to complex for 30-40 min, after which the
cells were transfected with the lipofectamine/cDNA complex for
4-6 h in serum-reduced OptiMEM (Gibco Laboratories) medium. For electrophysiology, cells at ~30-50% density were transfected with a total of 2 µg DNA per 35-mm dish. For biochemistry, cells at ~85-95% density were transfected with 10 µg per
60-mm dish (equal amounts of Kv1.3 cDNA and either EGFr or
control vector cDNA). Expression levels after 48-72 h allow the
recording of macroscopic currents from membrane patches and
detection of proteins by immunoprecipitation and Western blotting.
Biochemistry
Kv1.3 protein expression was measured using Western blot methods as described previously (Holmes et al., 1996a). Cells were harvested 2 d after transfection by lysis in ice-cold lysis buffer. Lysate proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose blots. These Western blots were then
probed with anti-Kv1.3 antiserum (Cai and Douglass, 1993
), and
bound antibody was visualized by enhanced chemiluminescence
(Amersham Corp., Arlington Heights, IL) to measure Kv1.3 protein expression. Film autoradiograms of Western blots were analyzed by densitometry using an imaging densitometer (GS-670;
Bio-Rad Laboratories, Richmond, CA).
Electrophysiology
Macroscopic currents were recorded at room temperature from
cell-attached membrane patches, using an Axopatch 200A (Axon
Instruments, Foster City, CA) patch clamp amplifier. To zero the
cell resting membrane potential, the bath solution contained
(mM): 150 KCl, 10 HEPES, 1 EGTA, 0.5 MgCl2, pH 7.5. The pipette solution contained (mM): 30 KCl, 120 NaCl, 2 CaCl2, 10 HEPES, pH 7.5. Pipettes (M15/10; Jencons Glass, Leighton Buzzard, UK) typically had resistances of 4-6 M, and were coated
near the tip with beeswax to reduce the capacitance of the glass.
The standard pulse protocol consisted of voltage steps to +40
mV for 1 s, at a frequency of one per minute to avoid cumulative
inactivation of Kv1.3 (Marom et al., 1993
). Before application of
any drugs, the current amplitude and inactivation rate were allowed to stabilize for 5 min after obtaining a seal. For drug treatment during patch recording, compounds were perfused directly
into the bath through 2-µl capillary tubes. The amplifier output
was filtered at 1-2 kHz, digitized at 2-5 kHz, and stored for later
analysis. Data were collected and stored using software written by
M. Bowlby and S. Marom, and hardware purchased from Microstar Laboratories, Inc. (Bellevue, WA). Inactivation of macroscopic current was fit to the sum of two exponentials by minimizing the sums of squares (Kupper et al., 1995
).
Mutagenesis
Standard techniques of site-directed mutagenesis using the polymerase chain reaction were used to mutate channel tyrosine residues to phenylalanines. Briefly, the general approach was to carry out two successive PCR reactions. The first uses the channel cDNA as the template, with one upstream nonmutagenic oligonucleotide primer, and a downstream mutagenic primer with a one- to three-base mismatch. The product of this first PCR reaction is used as the upstream primer for the second PCR reaction, with the downstream primer being nonmutagenic. The amplified mutant DNA fragment is cut sequentially with flanking restriction enzymes, purified and ligated into the channel backbone (after the equivalent wild-type region has been removed). All mutants were sequenced to confirm the presence of the mutation and to detect any sequence errors introduced during the PCR reactions. Six tyrosine residues (at four locations) in Kv1.3 lie within tyrosine kinase consensus phosphorylation sequences and were targeted for mutation.
Expression of Kv1.3
Expression of Kv1.3 in HEK 293 cells produces large
amounts of channel protein, thus allowing both the
biochemical detection of channels using Western blotting techniques and the recording of macroscopic currents from membrane patches (Bowlby and Levitan, 1995; Holmes et al., 1996a
). The expression of Kv1.3, as
measured on a Western blot, is not altered by coexpression with the EGFr, when compared with coexpression
of Kv1.3 and control vector (Fig. 1 A). Similarly, the expression of Kv1.3 is not affected by treatment with EGF
(Fig. 1 A) or insulin (Fig. 1 B) for at least 60 min, although insulin treatment for 4 h or longer does reduce
the expression of the channel (data not shown). Quantitative densitometric analysis of the Western blots (Table I) confirms these conclusions.
Table I. Effects of Receptor Expression and Activation on Kv1.3 Protein Expression |
Suppression of Kv1.3 Current by Activation of Growth Factor Receptors
Previous biochemical experiments have shown that
Kv1.3 is phosphorylated robustly on tyrosine residues
when the channel is coexpressed with the EGFr in HEK
293 cells (Holmes et al., 1996a). To determine whether
acute EGFr activation produces changes in the functional properties of Kv1.3, cotransfected cells were exposed to EGF during a patch recording. The amplitude
of Kv1.3 current in cell-attached patches is decreased
when 10 ng/ml EGF is perfused over the cell (Fig. 2 A).
The suppression by EGF begins within several minutes
and generally continues for as long as the patch survives, although some patches could be held sufficiently
long for a more-or-less steady state level of current to
be reached (Fig. 2 B). This suppression by EGF applied
during patch recording is blocked by addition of the
tyrosine kinase inhibitor erbstatin, indicating that tyrosine kinase activation is necessary for current suppression. Application of erbstatin alone for up to 1 h
has no effect (Fig. 2, B and C).
Longer term manipulation of EGFr activity also leads
to modulation of Kv1.3. When cells are cotransfected
with Kv1.3 and the EGFr, and exposed to EGF (10 ng/
ml) before patch recording, Kv1.3 current is decreased
compared with control cells transfected with Kv1.3 and
vector (Fig. 3 A). On average, this protocol of coexpression and EGF pretreatment decreases the current recorded in a cell-attached patch by approximately half
(Fig. 3 B), somewhat more than acute EGF treatment
(Fig. 2). To test the specificity of this effect, we blocked
EGFr activity with an EGFr antibody (-EGFr) that selectively inhibits binding of EGF to its receptor and
thus blocks subsequent receptor dimerization and kinase activation (Wu et al., 1989
). Pretreatment of cells
coexpressing Kv1.3 and the EGFr with
-EGFr for 4-12 h
more than reverses the decrease in current (Fig. 3 A),
while pretreatment with inactivated
-EGFr has no effect (Fig. 3 B).
It is noteworthy that the Kv1.3 current amplitude in
cotransfected cells exposed to -EGFr is more than six
times greater than that in cotransfected cells that were
exposed to EGF before recording (Fig. 3 B). In addition, the current in
-EGFr-treated cotransfected cells
is greater than in untreated cells transfected with Kv1.3
and control vector (Fig. 3 B). This result indicates that
an endogenous EGFr expressed in HEK 293 cells may
be partially suppressing current, even in the absence of
coexpressed EGFr or added EGF. Such endogenous
EGFr can be detected in HEK 293 cell lysates, separated
by SDS-PAGE/Western blots and probed with EGFr and
phosphotyrosine-specific antibodies (data not shown).
Insulin treatment also produces a decrease in peak
current amplitude in Kv1.3-transfected cells, presumably due to activation of an endogenous insulin receptor present in HEK 293 cells. Acute treatment of Kv1.3-transfected cells with 10 µg/ml insulin during a patch
recording suppresses current recorded in cell-attached patches by approximately half after 10 min of perfusion
(Fig. 4, A and C). Treatment of Kv1.3-transfected cells
with insulin for 5-18 h before patch recording elicits an
even larger decrease in current amplitude when compared with untreated Kv1.3-transfected cells (Fig. 4 B).
Although short term treatment with insulin does not affect channel expression (Fig. 1, Table I), it should be noted that insulin treatment for 4 h or longer reduces
Kv1.3 protein levels as detected by Western blots (data
not shown).
Modulation of C-Type Inactivation of Kv1.3
The kinetics of C-type inactivation of Kv1.3 are altered
markedly by EGFr coexpression and activation. When
cells cotransfected with Kv1.3 and the EGFr are treated
with EGF during patch recording, the inactivation rate
of the current increases (Fig. 5, A and B). No consistent
change in the activation or deactivation rates are found
(data not shown). The change in inactivation rate induced by EGF treatment is blocked when the cell is pretreated with the tyrosine kinase inhibitor erbstatin, and
then exposed to EGF together with erbstatin during the
patch recording (Fig. 5 C). Cells cotransfected with Kv1.3
and the EGFr, and pretreated with EGF (10 ng/ml) before patch recording, also show a faster rate of inactivation when compared with Kv1.3 and vector-transfected
controls (Fig. 6, A and B). Pretreatment of these cotransfected cells for 4-14 h with -EGFr reverses this effect,
and even slows the inactivation rate beyond that of the
control (Fig. 6, A and B). Thus, interruption of tyrosine
kinase activity or EGF binding blocks the EGFr-induced alteration of the inactivation rate.
In contrast, neither short (minutes) nor longer (up to
18 h) term insulin treatment of Kv1.3-transfected cells
has a significant effect on the rate of C-type inactivation
of the channel (Fig. 7, A and B). This difference in the
physiological effects of EGFr and insulin receptor stimulation implies that activation of the two receptor tyrosine kinases results in different patterns of phosphorylation of Kv1.3.
Two Distinct Actions of EGF on Kv1.3
The speeding of the inactivation rate observed with
EGF perfusion usually occurs simultaneously with the
decrease in current amplitude, although in several experiments the inactivation rate changed before the current amplitude decreased, or only the inactivation rate
increased. This led us to hypothesize that the two effects of EGF might result from distinct molecular
events. The Kv1.3 channel has six putatively internal tyrosine residues that reside in consensus sequences for
phosphorylation by tyrosine kinases (Songyang et al.,
1994). Each of these tyrosine residues was mutated to
phenylalanine and the mutant channels were tested for
their responses to EGF. Three of these tyrosines are adjacent to one another at positions 111, 112, and 113, and thus they were treated as a unit and mutated together (Y Y Y111-113FFF). Acute EGF treatment of cells
expressing the Kv1.3, Y137F, and Y449F mutant channels elicits a decrease in peak current and a speeding of
inactivation similar to that of the wild-type channel
(Fig. 8, A and B). A combination mutant of Y449F and
Y Y Y111-113FFF also behaves like the wild-type Kv1.3
with respect to its response to EGF (Fig. 8 C). In contrast, Kv1.3 Y479F does not exhibit a change in peak current in response to EGF, although the effect of EGF
on the inactivation rate is still intact (Fig. 8 D). The average EGF-induced inhibition of current is significant
(paired t test, P < 0.05) for all the mutant channels except for Kv1.3 Y479F (Fig. 8 E), while the increase in
the inactivation rate is significant (paired t test, P < 0.05) for all the mutant channels (Fig. 8 F). Thus, the
EGF-elicited changes in peak current and inactivation
rate are dissociable and are probably caused by different molecular mechanisms.
We have shown previously that expression and activation of an EGFr results in increased tyrosine phosphorylation of Kv1.3 in HEK 293 cells (Holmes et al.,
1996a). The present results demonstrate that coexpression of the EGFr and Kv1.3, followed by EGF treatment, produces a decrease in peak current levels and a speeding of C-type inactivation. Both of these changes are
blocked by pretreatment with
-EGFr, suggesting that
they require receptor dimerization and tyrosine kinase
activation. Insulin treatment produces a similar decrease
in peak current, but no change in C-type inactivation kinetics, suggesting that the actions of receptor tyrosine
kinases on current amplitude and inactivation kinetics
are distinct and separable. EGF treatment of cotransfected cells suppresses Kv1.3 current and speeds the inactivation within tens of minutes; both of these effects
are tyrosine kinase dependent because they are blocked
by a tyrosine kinase inhibitor. These two effects of EGF are also dissociable. Suppression of current by EGF is
dependent upon the presence of Y479 in the channel,
while the change in inactivation kinetics is not altered
by mutating any of the likely targets of tyrosine kinases.
Changes in Kv1.3 amplitude and kinetics induced by
the nonreceptor Src tyrosine kinase also can be attributed to phosphorylation of different tyrosine residues
(Fadool et al., 1997
). Interestingly, Y479 is not required
for the suppression of Kv1.3 current by Src (Fadool et
al., 1997
), suggesting that suppression can be achieved
by different molecular mechanisms.
It is clear that ion channels are targets of tyrosine kinases. Tyrosine phosphorylation has a variety of physiological effects on channels, including activation of calcium channels (Peppelenbosch et al., 1991, 1992
), potentiation of NMDA receptor-mediated glutamate currents
(Wang and Salter, 1994
), stabilization of the assembly of acetylcholine receptors (Huganir et al., 1984
; Wallace et al., 1991
; Wallace, 1995
), and both potentiation
and inhibition of potassium channel activity (Peppelenbosch et al., 1991
; Huang et al., 1993
; Lev et al., 1995
;
Holmes et al., 1996a
, 1996b
; Jonas et al., 1996
; Fadool
et al., 1997
). Modulation of ion channels by growth factor tyrosine kinases often occurs at the level of gene expression and protein synthesis, as several studies have reported effects on mRNA levels over several days (Fanger
et al., 1995
; Lesser and Lo, 1995
; Levine et al., 1995
).
Suppression of Kv1.5, however, occurs rapidly upon exposure of cells to platelet-derived growth factor or fibroblast growth factor (Timpe and Fantl, 1994
), and
similar acute exposure to EGF suppresses Kv1.2 (Peralta, 1995
). In both of these examples, PLC
activation
is required for suppression, although it is not known
whether phosphorylation (tyrosine or otherwise) of the
channel is involved.
Some of the effects of activation of the EGFr and the
insulin receptor on Kv1.3 current levels reported here
may be due either to a change in channel activity or to
a reduced number of channels on the cell surface, or
both. Growth factor receptors internalize soon after activation (reviewed by Schlessinger and Ullrich, 1992), and this conceivably might result in nonspecific internalization of other membrane proteins, such as ion
channels. The decrease in current observed with growth
factor activation could be due in whole or in part to
such internalization of the channel. Similarly, the reversal of current suppression by
-EGFr may reflect a
larger amount of channel protein on the cell surface.
These putative internalization effects may not be detectable on Western blots (unless internalization is coupled with protein degradation), as this method measures protein in all compartments of the cell. However,
the acute effects of EGF and insulin that occur during a
patch recording are probably due to phosphorylation
of the channel and not to internalization since: (a) the
cell-attached patch is not exposed directly to EGF or insulin and hence receptors in the patch, immediately
adjacent to the channels, do not bind ligand; and (b)
internalization of membrane from the patch probably would disrupt the patch and interrupt the recording.
The effects of EGF and insulin may not all be due to
direct phosphorylation of the channel by the receptors,
especially given the fact that the patch of membrane
containing the channels is isolated from direct contact
with the ligand by the recording electrode. The modulatory process therefore is likely to be dependent on intracellular factors in an endogenous signal transduction cascade. In the case of EGF, this cascade presumably results in phosphorylation of Y479, which causes
the decrease in current amplitude, although serine/
threonine protein kinases may also be involved in the
pathway. In contrast to the effects on peak current, the
change in inactivation kinetics induced by the EGFr may not be due to tyrosine phosphorylation of the
channel, as the inactivation change still occurs when
each of the tyrosine residues in good consensus sequences
for tyrosine kinases is mutated. Changes in inactivation
may be caused by phosphorylation of serine/threonine residues or other tyrosine residues, or by other non-
phosphorylation-dependent mechanisms (e.g., Bowlby
and Levitan, 1995).
The EGFr is expressed widely, and its activation is
critical for signaling in many cell types (Threadgill et
al., 1995), including neurons (Morrison et al., 1987
;
Kornblum et al., 1990
). Activation of the EGFr in overexpressing cell lines leads to activation of phospholipase A2, activation of a voltage-independent calcium channel, and calcium influx (Peppelenbosch et al.,
1991
, 1992
). This calcium influx leads, in turn, to activation of calcium-dependent potassium channels and
subsequent hyperpolarization of the cell resting potential (Pandiella et al., 1989
; Magni et al., 1991
). Fundamental aspects of cell physiology are affected greatly by such fluctuations in the resting membrane potential.
The modulation of Kv1.3 current by EGFr or insulin receptor activation will generally increase cell excitability
and oppose any hyperpolarization-induced decrease in
excitability. Thus, complex integration of signals at the
level of individual ion channels could lead to changes in growth rates, calcium influx, and a host of other cellular phenomena.
Address correspondence to Irwin B. Levitan, Volen Center for Complex Systems, Brandeis University, Waltham, MA 02254. Fax: 781-736-2339; E-mail: levitan{at}volen.brandeis.edu
Received for publication 27 May 1997 and accepted in revised form 21 August 1997.
Dr. Bowlby's current address is Wyeth-Ayerst Research, CNS Disorders, CN-8000, Princeton, NJ 08543-8000. Dr. Fadool's current address is Department of Zoology and Wildlife Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849-5414.The authors thank Kevin Berman for construction of the Kv1.3 Y449F mutant channel.
This work was supported by a research grant to I.B. Levitan, and National Research Service Awards from the National Institutes of Health to D.A. Fadool and T.C. Holmes.
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