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 (α-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 α-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 PLCγ, 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 β 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 MΩ, 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.

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 (α-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 α-EGFr for 4–12 h more than reverses the decrease in current (Fig. 3,A), while pretreatment with inactivated α-EGFr has no effect (Fig. 3 B).

It is noteworthy that the Kv1.3 current amplitude in cotransfected cells exposed to α-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 α-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 α-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 α-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, PLCγ 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 α-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.

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.

Bowlby
MR
,
Levitan
IB
Block of cloned voltage-gated potassium channels by the second messenger diacylglycerol independent of protein kinase C
J Neurophysiol
1995
73
2221
2229
[PubMed]
Cai
Y-C
,
Douglass
J
In vivo and in vitro phosphorylation of the T lymphocyte type n(Kv1.3) potassium channel
J Biol Chem
1993
268
23720
23727
[PubMed]
Chandy
KG
Simplified gene nomenclature
Nature (Lond)
1991
352
26
[PubMed]
Chandy
KG
,
Gutman
GA
Nomenclature for mammalian potassium channel genes
TIPS (Trends Pharmacol Sci)
1993
14
434
[PubMed]
Choi
KL
,
Aldrich
RW
,
Yellen
G
Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+channels
Proc Natl Acad Sci USA
1991
88
5092
5095
[PubMed]
Clark
S
,
Dunlop
M
Modulation of phospholipase A2activity by epidermal growth factor in CHO cells transfected with human EGF receptor
Biochem J
1991
272
715
721
[PubMed]
Dikic
I
,
Schlessinger
J
,
Lax
I
PC12 cells overexpressing the insulin receptor undergo insulin-dependent neuronal differentiation
Curr Biol
1994
4
702
708
[PubMed]
Fadool
DA
,
Holmes
TC
,
Berman
K
,
Dagan
D
,
Levitan
IB
Tyrosine phosphorylation modulates amplitude and kinetics of a neuronal voltage-gated potassium channel
J Neurophysiol
1997
78
1563
1573
[PubMed]
Fanger
GR
,
Jones
JR
,
Maue
RA
Differential regulation of neuronal sodium channel expression by endogenous and exogenous tyrosine kinase receptors expressed in rat pheochromocytoma cells
J Neurosci
1995
15
202
213
[PubMed]
Hanley
MR
Proto-oncogenes in the nervous system
Neuron
1988
1
175
182
[PubMed]
Hawley-Nelson
P
,
Ciccarone
V
,
Gebevehu
G
,
Jessee
J
A new polycationic liposome reagent with enhanced activity for transfection
FASEB J
1993
7
A167
Hille, B. 1992. Ionic Channels in Excitable Membranes. 2nd ed. Sinauer Associates, Inc., Sunderland, MA. 607 pp.
Holmes
TC
,
Fadool
DA
,
Levitan
IB
Tyrosine phosphorylation of the Kv1.3 potassium channel
J Neurosci
1996a
16
1581
1590
[PubMed]
Holmes
TC
,
Fadool
DA
,
Ren
R
,
Levitan
IB
Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain
Science (Wash DC)
1996b
274
2089
2091
[PubMed]
Hoshi
T
,
Zagotta
WN
,
Aldrich
RW
Biophysical and molecular mechanisms of Shakerpotassium channel inactivation
Science (Wash DC)
1990
250
533
538
[PubMed]
Huang
X-Y
,
Morielli
AD
,
Peralta
EG
Tyrosine kinase–dependent suppression of a potassium channel by the G protein-coupled m1 muscarinic acetylcholine receptor
Cell
1993
75
1145
1156
[PubMed]
Huganir
RL
,
Miles
K
,
Greengard
P
Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase
Proc Natl Acad Sci USA
1984
81
6968
6972
[PubMed]
Jonas
EA
,
Kaczmarek
LK
Regulation of potassium channels by protein kinases
Curr Opin Neurobiol
1996
6
318
323
[PubMed]
Jonas
EA
,
Knox
RJ
,
Kaczmarek
LK
,
Schwartz
JH
,
Solomon
DH
Insulin receptor in Aplysianeurons: characterization, molecular cloning, and modulation of ion currents
J Neurosci
1996
16
1645
1658
[PubMed]
Jonas
EA
,
Knox
RJ
,
Smith
TCM
,
Wayne
NL
,
Connor
JA
,
Kaczmarek
LK
Regulation by insulin of a unique neuronal Ca2+pool and of neuropeptide secretion
Nature (Lond)
1997
385
343
346
[PubMed]
Kornblum
HI
,
Raymon
HK
,
Morrrison
RS
,
Cavanaugh
KP
,
Bradshaw
RA
,
Leslie
FM
Epidermal growth factor and basic fibroblast growth factor: effects on an overlapping population of neocortical neurons in vitro
Brain Res
1990
535
255
263
[PubMed]
Kues
WA
,
Wunder
F
Heterogeneous expression patterns of mammalian potassium channel genes in developing and adult rat brain
Eur J Neurosci
1992
4
1296
1308
[PubMed]
Kupper
J
,
Bowlby
MR
,
Marom
S
,
Levitan
IB
Intracellular and extracellular amino acids that influence C-type inactivation and its modulation in a voltage-dependent potassium channel
Pflügers Arch
1995
430
1
11
[PubMed]
Lesser
SS
,
Lo
DC
Regulation of voltage-gated ion channels by NGF and ciliary neurotrophic factor in SK-N-SH neuroblastoma cells
J Neurosci
1995
15
253
261
[PubMed]
Lev
S
,
Moreno
H
,
Martinez
R
,
Canoll
P
,
Peles
E
,
Musacchio
JM
,
Plowman
GD
,
Rudy
B
,
Schlessinger
J
Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions
Nature (Lond)
1995
376
737
745
[PubMed]
Levine
ES
,
Dreyfus
CF
,
Black
IB
,
Plummer
MR
Differential effects of NGF and BDNF on voltage-gated calcium currents in embryonic basal forebrain neurons
J Neurosci
1995
15
3084
3091
[PubMed]
Levitan
IB
Modulation of ion channels by protein phosphorylation and dephosphorylation
Annu Rev Physiol
1994
56
193
212
[PubMed]
Lewis
RS
,
Cahalan
MD
Ion channels and signal transduction in lymphocytes
Annu Rev Physiol
1990
52
415
430
[PubMed]
Lewis
RS
,
Cahalan
MD
Potassium and calcium channels in lymphocytes
Annu Rev Immunol
1995
13
623
653
[PubMed]
Lopez-Barneo
J
,
Hoshi
T
,
Heinemann
SF
,
Aldrich
RW
Effects of external cations and mutations in the pore region on C-type inactivation of Shakerpotassium channels
Receptors Channels
1993
1
61
71
[PubMed]
Magni
M
,
Meldolesi
J
,
Pandiella
A
Ionic events induced by epidermal growth factor
J Biol Chem
1991
266
6329
6335
[PubMed]
Marom
S
,
Goldstein
SAN
,
Kupper
J
,
Levitan
IB
Mechanism and modulation of inactivation of the Kv3 potassium channel
Receptors Channels
1993
1
81
88
[PubMed]
Morrison
RS
,
Kornblum
HI
,
Leslie
FM
,
Bradshaw
RA
Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor
Science (Wash DC)
1987
238
72
75
[PubMed]
Pandiella
A
,
Magni
M
,
Lovisolo
D
,
Meldolesi
J
The effects of epidermal growth factor on membrane potential
J Biol Chem
1989
264
12914
12921
[PubMed]
Peppelenbosch
MP
,
Tertoolen
LGJ
,
de Laat
SW
Epidermal growth factor–activated calcium and potassium channels
J Biol Chem
1991
266
19938
19944
[PubMed]
Peppelenbosch
MP
,
Tertoolen
LGJ
,
den Hertog
J
,
de Latt
SW
Epidermal growth factor activates calcium channels by phospholipase A2/5-lipoxygenase-mediated leukotriene C4production
Cell
1992
69
295
303
[PubMed]
Peralta
EG
Dual modulation of a potassium channel by the m1 muscarinic and 2-adrenergic receptors
Life Sci
1995
56
957
964
[PubMed]
Rettig
J
,
Heinemann
SH
,
Wunder
G
,
Lorra
C
,
Parcej
DN
,
Dolly
JO
,
Pongs
O
Inactivation properties of voltage-gated K+channels altered by presence of β-subunit
Nature (Lond)
1994
369
289
294
[PubMed]
Schlessinger
J
,
Ullrich
A
Growth factor signaling by receptor tyrosine kinases
Neuron
1992
9
383
391
[PubMed]
Siegelbaum
SA
Ion channel control by tyrosine phosphorylation
Curr Biol
1994
4
242
245
[PubMed]
Songyang
Z
,
Blechner
S
,
Hoagland
N
,
Hoekstra
MF
,
Piwnica-Worms
H
,
Cantley
LC
Use of an oriented peptide library to determine the optimal substrates of protein kinases
Curr Biol
1994
4
973
982
[PubMed]
Threadgill
DW
,
Dlugosz
AA
,
Hansen
LA
,
Tennenbaum
T
,
Lichti
U
,
Yee
D
,
LaMantia
C
,
Mourton
T
,
Herrup
K
,
Harris
RC
et al
Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype
Science (Wash DC)
1995
269
230
233
[PubMed]
Timpe
LC
,
Fantl
WJ
Modulation of a voltage-activated potassium channel by peptide growth factor receptors
J Neurosci
1994
14
1195
1201
[PubMed]
Wallace
BG
,
Qu
Z
,
Huganir
RL
Agrin induces phosphorylation of the nicotinic acetylcholine receptor
Neuron
1991
6
869
878
[PubMed]
Wallace
BG
Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase
J Cell Biol
1995
128
1121
1129
[PubMed]
Wang
YT
,
Salter
MW
Regulation of NMDA receptors by tyrosine kinases and phosphatases
Nature (Lond)
1994
369
233
235
[PubMed]
Wilson
GF
,
Kaczmarek
LK
Mode-switching of a voltage-gated cation channel is mediated by a protein kinase A–regulated tyrosine phosphatase
Nature (Lond)
1993
366
433
438
[PubMed]
Wu
D
,
Wang
L
,
Sato
GH
,
West
KA
,
Harris
WR
,
Crabb
JW
,
Sato
JD
Human epidermal growth factor (EGF) receptor sequence recognized by EGF competitive monoclonal antibodies
J Biol Chem
1989
264
17469
17475
[PubMed]

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.

1

Abbreviations used in this paper: EGFr, epidermal growth factor receptor; Kv, voltage-dependent potassium.

Author notes

Address correspondence to Irwin B. Levitan, Volen Center for Complex Systems, Brandeis University, Waltham, MA 02254. Fax: 781-736-2339; E-mail: levitan@volen.brandeis.edu