A voltage-driven switch for ion-independent signaling by ether-à-go-go K⁺ channels

Transfection with EAG Stimulates Proliferation.

Fig. 1A shows a representative experiment demonstrating an increase in NIH 3T3 cell density after transfection with eag. Cell density was significantly higher for coverslips transfected with eag than for controls transfected with empty vector (P < 0.01; similar results obtained for two other experiments). To determine the mechanism underlying this increase, cells were labeled with BrdUrd, a marker for proliferation. Coverslips transfected with eag displayed substantial increases in BrdUrd incorporation when compared with vector-transfected controls (Fig. 1B) (P < 0.0001; n = 3). In contrast, transfection with the gene encoding Shaker, another voltage-dependent K⁺ channel, resulted in BrdUrd incorporation that was indistinguishable from control levels, indicating that the effect was specific to EAG. Increased proliferation also was observed by using phospho-histone labeling, another marker for proliferation (data not shown). These results indicate that proliferation accounts, at least in part, for the observed increase in cell density. EAG-induced proliferation was not limited to NIH 3T3 cells because EAG also increased proliferation in C2C12 myoblasts (data not shown). Finally, increased proliferation was also observed in response to EAG when cells were “synchronized” in serum-free media before reintroduction of FBS. However, proliferation was increased even in the complete absence of FBS (Fig. 1C) (P < 0.0001; n = 3); thus, the growth factors present in serum were not required for the effect of EAG on signaling.

EAG-Induced Proliferation Is Independent of Ion Flux.

K⁺ currents are essential for the proliferation of numerous cell types, including T lymphocytes and Schwann cells (15, 16). The role of K⁺ channels in proliferation, as well as other cellular processes, is generally assumed to be indirect. K⁺ channels alter the membrane potential to modulate Ca²⁺ influx through voltage-dependent Ca²⁺ channels, which, in turn, affects numerous intracellular messenger pathways (17, 18). However, in the present experiments, ion conduction was not required for the effect of EAG on proliferation. Fig. 2A shows recordings from Xenopus oocytes expressing wild-type EAG, myc-tagged EAG, or EAG-F456A, which contains a point mutation in the selectivity filter of the channel pore. The selectivity filter sequence is conserved in all K⁺ channels (19), and point mutations in this sequence eliminate conduction in Shaker, as well as numerous other K⁺ channels (8, 20, 21). Both myc-EAG and EAG-F456A failed to produce the outward currents characteristic of the wild-type channel. Comparison of current–voltage relationships (Fig. 2A Right) revealed little difference between myc-EAG and EAG-F456A currents and currents recorded from water-injected controls, which are carried by channels endogenous to oocytes. Although the mechanism underlying the absence of current in myc-EAG is unclear, both myc-EAG and EAG-F456A produced detectable gating currents (data not shown), indicating that defects in the folding or trafficking of EAG cannot wholly account for the absence of K⁺ current.

Myc-eag and eag-F456A increased proliferation to a degree similar to wild-type channels (Fig. 2B). The increases in BrdUrd incorporation were significant in comparisons with vector-transfected controls (P < 0.0001; n = 3). Moreover, the effects of myc-eag and eag-F456A were not significantly different from the effect of the wild-type channel. In short, changes in K⁺ flux, and the changes in membrane potential and Ca²⁺ influx that are presumed to result, cannot account for the proliferation induced by EAG. Additional evidence that the signaling mechanism of EAG does not include an indirect effect on Ca²⁺ influx was obtained by incubating cells in EGTA-buffered media before and during incubation of cells with BrdUrd. EAG-induced proliferation in the presence of EGTA (1 mM, 5 h) was 90.1 ± 11.2% and 95.1 ± 3.7% of the proliferation in standard Ca²⁺-containing media for wild-type EAG and EAG-F456A channels, respectively (n = 3; not significant). Higher concentrations of EGTA caused cells to detach and, therefore, were not assessed.

EAG-Induced Proliferation Requires the p38 Mitogen-Activated Protein (MAP) Kinase Pathway.

MAP kinase signaling is central to proliferation in numerous cell types and in response to a variety of signals (22). To determine whether the proliferation induced by nonconducting EAG channels requires this pathway, cells were treated with inhibitors of MAP kinase signaling in serum-free media. The p38 MAP kinase inhibitor SB-203580 [4-(4-fluorophenyl)-2-(4-methylsufinylphenyl)-5-(4-pyridyl)-1H-imidazole; Calbiochem] (20 μM) blocked the proliferation observed in response to EAG-F456A (Fig. 2C) (P < 0.0001; n = 3), reducing proliferation to levels that were no different from the proliferation observed for controls (P > 0.05; n = 3). Similar results were obtained by using the “p38 MAP kinase inhibitor” [2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one; Calbiochem; Fig. 2D Left]. In contrast, although the control compound SB-202474 [4-ethyl-2(p-methoxyphenyl)-5-(4′-pyridyl)-1H-imidazole; Calbiochem] reduced the overall level of proliferation in both vector- and eag-F456-transfected conditions, it failed to inhibit the EAG-specific increase (Fig. 2D Center) (P < 0.0001; n = 2). Finally, although PD-98059 (2′-amino-3′-methoxyyflavone; Calbiochem), an inhibitor of the p44/42 extracellular signal-regulated kinases, reduced proliferation in the presence of FBS (data not shown), PD-98059 (40 μM) had little effect on the increase in proliferation specifically induced by nonconducting EAG in serum-free media (Fig. 2D Right) (P < 0.01; n = 3). These results suggest that p38, but not p44/42, MAP kinase signaling is required for the proliferation stimulated by nonconducting EAG-F456A channels.

To determine whether EAG affects p38 MAP kinase activity, we immunoblotted NIH 3T3 cell lysates with antibodies that detect either total p38 MAP kinase or, specifically, the phosphorylated, active kinase. As shown in Fig. 2E, p38 phosphorylation nearly doubled in the presence of either wild-type or nonconducting EAG (Fig. 2E) (P < 0.05; n = 4), and the magnitude of the effect appeared to approximate the average increase in BrdUrd incorporation (Fig. 2 B and C).

EAG-Induced Proliferation Is Regulated by the Position of the Voltage Sensor.

The observation that the signaling activity of EAG does not depend on ion conduction predicts that changes in extracellular K⁺ concentration ([K⁺]_o) should not affect EAG-induced proliferation. However, although increased [K⁺]_o increased proliferation in vector-transfected controls, increasing [K⁺]_o by 10 mM inhibited EAG-induced proliferation, returning proliferation to control levels. Specifically, at 15 mM [K⁺]_o, EAG-induced proliferation was 93.9 ± 1.5% of controls compared with 151.4 ± 7.3% in normal 5.3 mM [K⁺]_o. [Measurements were normalized to vector-transfected controls in 5.3 mM (P < 0.001)]. Similar results were observed in two additional experiments. Because increases in [K⁺]_o will depolarize the membrane and shift the position of the voltage sensor even in nonconducting EAG channels, we hypothesized that the signaling activity of EAG might depend on voltage-sensitive conformations of the channel. Specifically, the [K⁺]_o experiments predict that increases in the proportion of channels in the open state should decrease EAG signaling activity.

To explore the possibility that the signaling activity of EAG might be regulated by the position of the voltage sensor, we examined the effects of EAG channels containing mutations in the sixth transmembrane segment that shifted their voltage dependence of activation. Fig. 3A shows representative currents obtained for the wild-type channel and two mutants, EAG-TATSSA (T449S/K460S/T470A) and EAG-HTEE (H487E/T490E), when expressed in Xenopus oocytes. Comparison of the conductance–voltage (G–V) relationships (Fig. 3B) reveals that the predominant effect of both mutations was to produce hyperpolarizing shifts in the midpoints of activation from 8.0 ± 1.1 mV (wild type, n = 6) to −10.8 ± 1.2 mV (n = 9) and −31.6 ± 2.0 mV (n = 7) for EAG-TATSSA and EAG-HTEE channels, respectively. In addition, the TATSSA and HTEE mutations also produced changes in kinetics; however, these changes were in opposite directions (Fig. 3A). Comparison of the average resting potentials of oocytes expressing EAG channels (Fig. 3C) revealed that the resting potentials closely followed the changes in the V₁₀ for activation (the voltage at which 10% of channels are activated) (Fig. 3D). This would be expected if EAG is the major channel contributing to the membrane potential. Wild-type EAG produced only a small shift in the resting potential from −44.5 ± 2.8 to −52.8 ± 1.9 mV. In contrast, EAG-TATSSA and EAG-HTEE shifted the resting potential to −82.8 ± 1.1 and −90.7 ± 0.9 mV, respectively. It is important to note that, given that K⁺ channel conformation and membrane potential act as a negative feedback loop, the proportion of channels in the closed state should be similar in each case, provided that each of the constructs contributes to the membrane potential to a similar extent.

The above EAG constructs were used to address whether the signaling activity of EAG is regulated by channel conformation. As shown in Fig. 4A, there was an ≈2-fold increase in the proliferation of NIH 3T3 cells regardless of whether wild-type eag, eag-TATSSA, or eag-HTEE were used. Given the negative feedback of conducting EAG channels on channel conformation, these results suggest that proliferation depends on the position of the voltage sensor of EAG channels rather than on a specific resting membrane potential. Indeed, measurement of the resting potentials of NIH 3T3 cells transfected with these constructs indicated that, although EAG channels contributed to the membrane potential to a lesser degree than in oocytes, each construct shifted the resting potential closer to the respective activation threshold (Fig. 4B).

Additional evidence in support of the hypothesis that channel conformation is a “switch” for the signaling activity of EAG was obtained by using eag-TATSSA and eag-HTEE constructs that had been rendered nonconducting by including the F456A mutation, in effect short-circuiting the negative feedback function of EAG. As expected, the resting potentials of cells expressing these double mutants were similar to the resting potentials of vector-transfected controls (Fig. 4B). The shifted voltage dependence of these channels, combined with their inability to shift the resting potential, should result in a larger proportion of TATSSA-F456A and HTEE-F456A channels in the open conformation. On the basis of the prediction above, increasing the proportion of channels in the open state should decrease EAG-induced proliferation. As predicted, both EAG-TATSSA/F456A and EAG-HTEE/F456A failed to increase proliferation above control levels (Fig. 4A). In contrast, as observed in our previous experiments, proliferation was robust for EAG-F456A. Finally, neither changes in expression level nor changes in the surface expression of EAG (Fig. 4C) could account for the changes in proliferation observed with the double-mutant constructs. Together, these results suggest that the signaling activity of EAG depends on voltage-sensitive conformations of the channel.

Plasmids and Construction.

pCS2-myc-eag contains a myc tag added to the amino terminus (26). For the wild-type construct, EcoRI and XbaI sites flanking the coding sequence were used to subclone eag (without the myc tag) into the pCS2 vector. A Kozak sequence (GCCACC) was added to improve channel expression. Shaker was subcloned into pCS2 by using EcoRI sites flanking the coding sequence of pGH19-Shaker (38). Eag-F456A, eag-H487E/T490E (HTEE), eag-T449S/A460S/T470A (TATSSA), and double mutants were generated by site-directed mutagenesis using QuikChange (Stratagene). For oocyte expression, mutant constructs were subcloned into pGH19-eag (39). All constructs were verified by sequencing.

Immunocytochemistry and Proliferation Assays.

NIH 3T3 fibroblasts were maintained at 37°C and 5% CO₂ in DMEM (Invitrogen) supplemented with 10% FBS as described (38). For transfection, coverslips were washed with Opti-MEM and incubated for 8–10 h in 350 μl of Opti-MEM containing 0.4 μl of the indicated cDNAs and 1.5 μl of Lipofectamine (Invitrogen). Coverslips were then washed and incubated in standard media for 12 h. For serum-free experiments, this was followed by incubation in FBS-free DMEM for 12 h, with 1% FBS added to a subset of wells as a positive control. MAP kinase inhibitors or control compounds were added after washout of Lipofectamine.

For BrdUrd labeling, 10 μM BrdUrd was added to each well for ≈60 min. Coverslips were washed with PBS and fixed with a 3:7 mixture of 50 mM glycine (pH 2.0)/100% ethanol for 1 h at room temperature and then denatured with 4 M HCl for 15 min. Cells were labeled with anti-BrdUrd fluorescein-conjugated antibody (Invitrogen) for 45 min at 37°C. Labeling was visualized by using an Olympus (New Hyde Park, NY) BX51W1 microscope equipped with a Qimaging Retiga Exi camera and iplab 3.6 software. Coverslips from the same experiment were viewed by using identical settings, and multiple representative scans were taken for each coverslip. To quantify fluorescence, scans were background subtracted, and the intensity of all pixels above background was summed across the total area of each scan. Total intensities were averaged across all scans for each condition before normalizing to the average intensity for controls. Normalized data were then averaged across experiments (n). Data were analyzed by using a two-way ANOVA with Tukey’s post hoc analysis with the condition and experiment number as variables (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.0001; N.S., not significant). Data are presented as the mean ± SEM. MAP kinase inhibitors were purchased from Calbiochem.

Biochemistry.

Cells grown on culture plates were dissociated with trypsin-EDTA 48 h after transfection, washed, and resuspended in PBS. Biotinylation and precipitation of EAG was performed as described (28). Briefly, cell suspensions were incubated in 2 mM sulfo-NHS-LC-biotin (Pierce), and the reaction was quenched by washing with 100 mM glycine in PBS. Cells were lysed in PBS supplemented with 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM DTT, and protease inhibitors, and the homogenate was centrifuged twice for 10 min at 20,000 × g. Protein concentrations of supernatants were determined by Bradford assay and diluted to ≈0.5 mg/ml. Surface proteins were precipitated with streptavidin agarose, and the precipitate was washed extensively before addition of sample loading buffer. Blots were probed with antisera directed against the carboxyl-terminal domain of EAG [EAG (CT) antisera; 1:2,000] in blocking buffer, followed by horseradish peroxidase-conjugated secondary antibody (1:2,000), and visualized with ECL (Amersham Biosciences). Protein bands were quantified by using quantity one software (Bio-Rad). Assays of p38 MAP kinase activity were performed by using extracts prepared in buffer containing the following: 20 mM Tris, 100 mM NaCl, 50 mM NaF, 1 mM Na₃V0₄, 1 mM EDTA, 1 mM DTT, 1 mM benzamidine, and 0.001 mM microcystin-LR with 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors.

Electrophysiology.

Experiments using Xenopus oocytes were performed as described (28). Oocytes were typically injected with 0.1–0.2 ng of RNA; nonconducting constructs were injected at 1–2 ng. The recording solution contained the following: 140 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 10 mM Hepes (pH 7.1 with NaOH). Pipettes had resistances of 0.3–0.6 MΩ. Experiments were performed at room temperature. Leak and capacitative currents were subtracted by using P/4 methods.

For NIH 3T3 cell recordings with an Axopatch200B amplifier (Molecular Devices), cells maintained on plates were cotransfected with pCDNA3-EGFP and the indicated constructs and were then replated onto coverslips. Pipette resistances ranged from 3 to 6 MΩ. The extracellular solution contained the following: 40 mM sodium aspartate, 100 mM NaCl, 4 mM KCl, 1.5 mM MgCl₂, 1 mM CaCl₂, 2 mM glucose, and 10 mM Hepes (pH 7.4 with NaOH). The pipette solution contained the following: 35 mM potassium aspartate, 110 mM KCl, 2 mM MgATP, 1 mM NaATP, 3 mM sodium phosphocreatine, 0.1 mM NaGTP, 8 mM EGTA, and 10 mM Hepes (pH 7.4 with KOH).