Effects of Estrogen on Neuronal KCNQ2/3 Channels Expressed in PC-12 Cells

Abstract

We previously reported that 17β-estradiol (E2) improves long term potentiation (LTP) in hippocampal neurons after global ischemia in rat. In the present study, we investigated if E2 can directly modulate the activity of neuronal KCNQ2/3 channels, the molecular entity of neuronal M-current in hippocampus, expressed in the PC-12 cells. We found that exogenous E2 inhibits the KCNQ2/3 channels in a dose-dependent fashion. The minimal inhibitory concentration of E2 is 10 µM. At testing membrane potential of +90 mV, the whole cell current density was reduced to 56.5, 49.3 and 31.9% of the control by 50, 20 and 10 µM of E2, respectively. The voltage-dependency of the KCNQ2/3 currents was also affected. E2 at 10, 20 and 50 µM shifted the half maximal activation voltage (V_1/2) from 13.8±2.3 mV (n=12) to 20.6±1.9 mV (n=8, p<0.05), 26.0±1.9 mV (n=8, p<0.001) and 27.6±3.5 mV (n=8, p<0.001), respectively. Our data indicate that exogenous E2 can directly affect the activity of KCNQ2/3 channels at pharmacological levels via a non-genomic pathway.

Estrogen (17β-estradiol, E2) exerts regulatory effects in a variety of tissues, including the brain.^1–4) The roles of estrogen in the brain are diverse and mostly beneficial, such as neuroprotection and cognition, therefore of considerable importance. More intriguingly, estrogen may delay the onset or ameliorate the severity of neurological disorders of Alzheimer’s disease, Parkinson’s disease, and stroke.^4–7)

Estrogen works via genomic and/or non-genomic signaling pathways. The genomic signaling pathway of estrogen involves receptor binding in cytosol and transcriptional effects in nucleus. On the other hand, studies have convincingly shown that estrogen induces rapid responses that are independent of nuclear localization and transcriptional regulations, especially in ion channel activities. Valverde et al. demonstrated that E2 activated the human Maxi-K channels (hSlo) by binding to β subunits of the channel complex.⁸⁾ Kakusaka et al. reported that E2 in physiological concentrations partially down-regulate the human cardiac rapidly-activating delayed rectifier K⁺ channels (human Ether-à-go-go Related Gene (hERG) channels) via non-genomic signaling pathways.⁹⁾ The mechanisms underlying rapid biological responses to estrogen may vary from case to case.

KCNQ2 and KCNQ3 are decoded from independent genes and belong to the KCNQ potassium channel subfamily. They presumably co-assemble in a ratio of 2 : 2 to form the neuronal KCNQ2/3 channels and are the molecular entity of M-currents in nervous system playing crucial roles in neurophysiology and diseases.^9,10) In hippocampus, KCNQ2/3 channels are colocalized to the axon initial segment (AIS) implicating its role in action potential generation.¹¹⁾ Functional studies further supported this notion by that the suppression of M-currents shifted the firing mode of hippocampal CA1 neurons from regular to burst by augmenting spikes after-depolarization, as well as by reducing the intrinsic subthreshold theta resonance.¹²⁾ Behavioral study also showed that KCNQ channel inhibitor XE991 could revert the cognitive impairment in a mice model.¹³⁾ Overall, evidences accumulated through recent researches strongly suggest the direct involvement of KCNQ2/3 channels in neuronal excitability.

Previously, we reported that E2 could improve the long-term potentiation (LTP) in hippocampal neurons of a mice stroke model.⁷⁾ Since KCNQ2/3 channels has been proposed as a “clamper” of the membrane potential in neurons, we hypothesized that KCNQ2/3 channels might be the key molecule mediating E2-induced changes of LTP in our previous findings. This study was designed to investigate whether E2 could directly regulate the activity of KCNQ2/3 channels in PC12 cells. Indeed, we found that whole-cell currents of KCNQ2/3 channels were significantly reduced with E2 in a dose-dependent manner. The membrane potentials of half-maximal voltage activation for KCNQ2/3 channels were also shifted to more depolarized values in the presence of E2. Taken together, our data suggest that E2 could regulate neuronal excitability via inhibiting the KCNQ2/3 channel activity.

Materials and Methods

Cell Culture and Heteroexpression of KCNQ2/3 Channels in PC-12 Cells

PC-12 cells were maintained in a humidified CO₂-incubator at 37°C. The media were Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT, U.S.A.). For heteroexpression of KCNQ2/3 channels, the cells were subcultured on glass coverslips of 5×5 mm for overnight, then co-transfected with hKCNQ2-pCDNA3.1, rKCNQ3-pCDNA3.1 (gifts from Prof. H Zhang, China) and green fluorescent protein (GFP)-pCDNA (Clontech, U.S.A.) plasmids in a ratio of 4 : 4 : 1 with Fugene HD (Roche, Switzerland). The GFP here served as the indication for successful transfection. The efficiency of transfection was about 20%.

Whole-Cell Patch Clamp Recording

Whole-cell patch mode was used to record the KCNQ2/3 channel currents from PC-12 cells 24–30h after transient transfection. Briefly, coverslips with PC-12 cells were placed in a recording chamber (0.5 mL in volume) mounted on an upright fluorescent microscope (Olympus BX51XI, Japan) and superfused with physiological saline at 2 mL·min⁻¹ at room temperature (25–27°C). Patch pipettes were pulled from borosilicate glass capillaries with a horizontal puller (Sutter Instruments P-97, Novato, CA, U.S.A.) and fire-polished to final resistances of 2–6 MΩ when filled with a pipette solution. Conventional whole cell recording was established following the formation of high resistance seal of 1–3 GΩ between pipette and PC-12 cells. Liquid junction potentials, fast and slow capacitance and serial resistance were compensated online with the acquisition software Patch Master (HEKA, Germany). Whole-cell currents were amplified using an EPC-10 amplifier (HEKA, Germany), sampled at 20 kHz, low-pass filtered at 5 kHz and stored onto a hard disk of an operating personal computer through a digital interface ITC-16 (HEKA, Germany). Voltage-gated KCNQ2/3 currents were induced from holding voltage of −80 mV with a family of voltage steps from −110 mV to +90 mV in 20 mV increments (Fig. 1).

Fig. 1. Whole Cell Currents of KCNQ2, KCNQ3 and KCNQ2/3 Channels Expressed in PC-12 Cells

Typical whole-cell currents were displayed with the stimulation voltage protocol.

Chemicals and Solutions

17β-Estradiol (E2) was purchased from Sigma and pre-resolved with dimethyl sulfoxide (DMSO) in a stock solution of 25 mM and kept in −20°C. The final concentrations of E2 were directly diluted from the E2 stock solution with physiological saline just before each experiment. The final DMSO concentration in perfusion solution was lower than 0.1% and confirmed with no significant effects on KCNQ2/3 currents (data not shown). The physiological saline contained (in mM): 140 NaCl, 5.4 KCl, 1 MgCl₂, 2 CaCl₂, 10 glucose, 10 N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.4). The pipette solution contained (in mM): 140 KCl, 1 MgCl₂, 10 ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 HEPES, 5K₂-ATP, pH 7.4. All of the above chemicals were obtained from Sigma.

Data Analysis and Statistics

The membrane capacitance of individual PC-12 cell was measured with a cancellation circuitry of the patch-clamp amplifier and stored with data files. Current densities (pA/pF) of KCNQ2/3 channels were subsequently calculated by dividing the tail currents with cell membrane capacitance and used for constructing the I–V curves for comparison.

The voltage–activation curves of KCNQ2/3 channels were derived from the I–V curves and fitted with the Boltzmann equation: G=(G_max−G_min)/(1+exp[(V−V_1/2)/S])+G_min, where G_max is the maximal membrane conductance, G_min is the minimal membrane conductance, V_1/2 is the membrane voltage for half maximal activation and S is the slope factor.

All data were processed with the Origin 8.0 software (Origin Lab, U.S.A.) and expressed as mean±S.E. Statistical significance between presence and absence of E2 was computed using one-way ANOVA, followed by Fisher’s least significant difference (LSD) test. p values less than 0.05 were considered to reflect a statistically significant difference.

Results

Whole-cell currents of KCNQ2, KCNQ3 and KCNQ2/3 channels were recorded from transient transfected PC-12 cells with the co-transfected GFP as an indicator. Similar to previous reports by other groups, large slowly activating, voltage-dependent outward currents were observed in PC-12 cells co-transfected with KCNQ2 and KCNQ3 while smaller currents were seen in the cells with KCNQ2 alone. In contrast, PC-12 cells expressing KCNQ3 channels showed only background levels of whole-cell currents, not significant from that seen in untransfected control cells (Fig. 1). These data validate that KCNQ2 and KCNQ3 form functional heteromers of KCNQ2/3 channels when co-transfected in PC-12 cells.¹⁴⁾

E2 led to a reduction in KCNQ2/3 channel currents at every stimulation voltage in a dose-dependent manner (Figs. 2A, 2B). E2 at 10, 20, 50 µM markedly decreased KCNQ2/3 current densities at +90 mV (56.5%, 49.3% and 31.9%, respectively) when compared to that of DMSO vehicle application (Fig. 2C). The onset time of E2 at 50 µM on KCNQ2/3 channels was less than 10 min after application (Fig. 3). The inhibitory effects of E2 on KCNQ2/3 channel activity were not recovered upon 10 min of wash-out (53.4%).

Fig. 2. Effects of E2 on KCNQ2/3 Channel Currents

Whole cell currents (2A), I–V curves (2B) and current densities at 90 mV (2C) of KCNQ2/3 channels were displayed in the absence or presence of different concentration of E2. ***: p<0.001.

Fig. 3. The Time Courses of KCNQ2/3 Currents Responding to E2

Current amplitude of each cell at 90 mV was normalized to its pre-E2 value. KCNQ2/3 currents were stable during 25 min of recording in the absence of E2 (n=13), and decreased 5 min after application of 50 µM E2 (n=5).

The half maximal voltage activation (V_1/2) of KCNQ2/3 channel currents was derived from the I–V curve. In the absence of E2, KCNQ2/3 currents displayed an averaged V_1/2 of 13.8±2.3 mV (n=12). Although 2 µM E2 did not make any significant shift of V_1/2 (15.6±1.2 mV, n=8, p>0.05), higher concentrations of E2 at 10, 20 and 50 µM did shift the V_1/2 to 20.6±1.9 mV (n=8, p<0.05), 26.0±1.9 mV (n=8, p<0.001) and 27.6±3.5 mV (n=8, p<0.001), respectively (Fig. 4). The slope factor S, which mostly reflects the dynamic gating characteristics of ion channels, of KCNQ2/3 channels in the absence of E2 was 20.8±1.56 mV (n=12), and similar values were observed after E2 administration at concentrations of 2, 10, 20 and 50 µM (19.8±1.2, 20.3±0.8, 21.5±1.1, 20.2±1.3 mV, respectively. n=8, p>0.05).

Fig. 4. Effect of E2 on the Voltage-Dependency of KCNQ2/3 Channels

E2 at 10, 20 and 50 µM significantly shifted V_1⁄2 from 15.6±1.2 mV to more positive values of 20.6±1.9 (p<0.05), 26.0±1.9 (p<0.001) and 27.6±3.5 mV (p<0.001), respectively.

Discussion

Our data presented in this study demonstrate that exogenous E2 inhibits the activity of neuronal KCNQ2/3 channels expressed in PC-12 cells. The inhibitory effects of E2 on KCNQ2/3 channels were dose-dependent, affecting both current density and voltage-dependency at concentrations as low as 10 µM. Although the effective concentrations of E2 in our study are much higher than the physiological levels of E2 in the circulation, it is still well within the therapeutic range seen in literature,¹⁴⁾ indicating that exogenous E2 can affect the function of KCNQ2/3 channels at pharmacological levels.

The mechanism underlying the inhibitory effects of E2 on KCNQ2/3 channels is not completely clear from the results of the present study. Based on the data presented here, the minimal concentration of E2 (10 µM) for effective inhibition is much higher for a specific binding between E2 and estrogen receptors (ERs) (K_d=0.05–0.1 nM).^15,16) Thus, it is unlikely that the inhibitory effects of exogenous E2 on KCNQ2/3 channels are due to ERs-mediated specific effects, although expression of all three ERs can be induced by nerve growth factor in the PC-12 cells.¹⁷⁾ In oocytes or HEK cells, currents mediated by heterologously expressed KCNQ2/3 channels were enhanced by activation of the cAMP-dependent protein kinase A (PKA) and this effect was abolished by site-directed mutations that eliminated a single PKA consensus site near the amino terminus of the KCNQ2 polypeptide.¹⁸⁾ We investigated the effect of E2 on cAMP in PC-12 cells, and cAMP levels were not significantly changed after exposure to E2 (data not shown). Whether E2 affects PKA activities was not investigated in this study. Since the effects of E2 on KCNQ2/3 channels in this report are quick and irreversible, it is more likely to be mediated via a non-genomic pathway. One possible mechanism is that the fast inhibitory effects of E2 are by means of E2 specifically binding to the KCNQ2/3 channel protein. An example for that has been seen in the estrogen-dependent activation of human Maxi-K channels.⁸⁾ Thus, our data suggest that KCNQ2/3 channels are pharmacologically modulated by exogenous E2. In addition, whether the modulation of E2 on the KCNQ2/3 channels is specific is still a question to be further investigated.

The importance of neuronal KCNQ2/3 channels in hippocampal plasticity has been clearly indicated in literatures. Results from immunohistochemistry study have shown that KCNQ2/3 channels are localized to the axon initial segment (AIS) in hippocampus.¹⁹⁾ Electrophysiological recordings have demonstrated the functional involvement of KCNQ2/3 channels in hippocampal plasticity. Application or injection of KCNQ2/KCNQ3 channel blockers lowered the threshold for LTP induction^20,21) and reversed the cognitive impairment in animal models of cognitive disease.¹³⁾ KCNQ2/KCNQ3 channel inhibition might be a key component in the enhancement of LTP and the improvement of memory function.²⁰⁾ Thus, our data in this study are for the first time to show that exogenous E2 exerts direct inhibitory effects on the KCNQ2/3 channels, suggesting an alternative mechanism for exogenous E2 on hippocampal plasticity. Since the results of this study were obtained under in vitro conditions, its significance in hippocampal plasticity needs to be verified under in vivo conditions.

Acknowledgment

This work was supported in part by Grants from the National Natural Science Foundation of China (30770573), the 973 program from the Minister of Science and Technology in China (2007CB512304) and the SRF for ROCS, SEM (2008–101). We are grateful to Professor H. L. Zhang of Hebei Medical University, who kindly provided the hKCNQ2-pCDNA3.1 and rKCNQ3-pCDNA3.1 plasmids.

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