Ischemic stroke is often accompanied by neuronal hyperexcitability (i.e., seizures), which aggravates brain damage. Therefore, suppressing stroke-induced hyperexcitability and associated excitoxicity is a major focus of treatment for ischemic insults. Both ATP-dependent and Ca2+-activated K+ channels have been implicated in protective mechanisms to suppress ischemia-induced hyperexcitability. Here we provide evidence that the localization and function of Kv2.1, the major somatodendritic delayed rectifier voltage-dependent K+ channel in central neurons, is regulated by hypoxia/ischemia-induced changes in metabolic state and intracellular Ca2+ levels. Hypoxia/ischemia in rat brain induced a dramatic dephosphorylation of Kv2.1 and the translocation of surface Kv2.1 from clusters to a uniform localization. In cultured rat hippocampal neurons, chemical ischemia (CI) elicited a similar dephosphorylation and translocation of Kv2.1. These events were reversible and were mediated by Ca2+ release from intracellular stores and calcineurin-mediated Kv2.1 dephosphorylation. CI also induced a hyperpolarizing shift in the voltage-dependent activation of neuronal delayed rectifier currents (IK), leading to enhanced IK and suppressed neuronal excitability. The IK blocker tetraethylammonium reversed the ischemia-induced suppression of excitability and aggravated ischemic neuronal damage. Our results show that Kv2.1 can act as a novel Ca2+- and metabolic state-sensitive K+ channel and suggest that dynamic modulation of IK/Kv2.1 in response to hypoxia/ischemia suppresses neuronal excitability and could confer neuroprotection in response to brief ischemic insults.
Hypoxia is the most common cause of neonatal seizures and a risk factor for the later development of epilepsy. In adults, ischemic attack is often accompanied by seizures (Varelas and Mirski, 2001), and the resulting hyperexcitability may aggravate brain damage from ischemic stroke. Indeed, there are many similarities between ischemia and epilepsy regarding brain-damaging mechanisms (Leker and Neufeld, 2003). In each of these pathological conditions, an acute membrane depolarization caused by voltage- and ligand-gated ion channels results in neuronal hyperexcitability, intracellular Ca2+ ([Ca2+]i) overload, and neuronal damage, together referred to as excitotoxicity (Lee et al., 1999). On the basis of these similarities, antiepileptic drugs have been tested as neuroprotective agents in animal models of stroke, and some, especially channel blockers, have shown promising effects (Calabresi et al., 2003). Therefore, suppressing neuronal hyperexcitability after ischemic insults might be a fertile therapeutic target for stroke.
Voltage-dependent K+ (Kv) channels are potent suppressors of neuronal excitability and represent key candidates for physiological and therapeutic modulation of the neuronal hyperexcitability that occurs in hypoxic/ischemic and epileptic patients. Delayed rectifier Kv currents (IK) are important in regulating somatodendritic excitability in hippocampal and cortical pyramidal neurons (Bekkers, 2000; Du et al., 2000; Korngreen and Sakmann, 2000). In these neurons, the Kv2.1 channel is the major component of somatodendritic IK (Martina et al., 1998; Murakoshi and Trimmer, 1999; Du et al., 2000; Malin and Nerbonne, 2002; Pal et al., 2003). Interestingly, many studies of somatodendritic IK, and of Kv2.1, in pyramidal neurons suggest a function in regulating excitability and Ca2+ influx during periods of repetitive high-frequency firing (Colbert and Pan, 1999; Bekkers, 2000; Du et al., 2000; Kang et al., 2000; Korngreen and Sakmann, 2000; Misonou et al., 2004) rather than in a more classical role in action potential repolarization. We found previously that increased neuronal activity induced by seizures or glutamate stimulation changes Kv2.1 phosphorylation state and localization and yields increased amplitude of hippocampal neuron IK/Kv2.1 attributable to hyperpolarizing shifts in voltage-dependent activation (Misonou et al., 2004). Such enhanced activity of Kv2.1 under conditions of hyperexcitability is predicted to provide homeostatic suppression of neuronal activity (Surmeier and Foehring, 2004; Misonou et al., 2005).
We present here the surprising finding that in vivo, brief CO2 anesthesia results in dephosphorylation of brain Kv2.1 and dispersion of somatodendritic Kv2.1 clusters in neurons throughout the brain. Short periods of hypoxia/ischemia can induce similar modulation of Kv2.1 channels in cultured hippocampal neurons by inducing intracellular Ca2+ release, presumably from mitochondria, and calcineurin-mediated Kv2.1 dephosphorylation. The ATP- and Ca2+-mediated modulation of Kv2.1 is associated with increased amplitude of neuronal IK attributable to a hyperpolarizing shift in the voltage dependence of activation and a hypoxia/ischemia-induced suppression of neuronal excitability. Together with our previous findings of Kv2.1 modulation in seizure animals (Misonou et al., 2004), we suggest that phosphorylation-dependent modulation of the abundant somatodendritic Kv2.1 channel may be a general mechanism to suppress pathological hyperexcitability of central neurons.
Materials and Methods
Preparation of hypoxic/ischemic animals. All animal use procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals described by the National Institutes of Health. Rats were subjected to CO2 inhalation in a closed chamber connected to a CO2 gas cylinder. Some animals were anesthetized by inhalation of diethyl ether or isoflurane in a closed chamber or by intraperitoneal injection of pentobarbital (50 mg/kg) or a mixture of ketamine and xylazine. Animals were then either decapitated to remove the brains or perfused with 4% paraformaldehyde for immunohistochemistry. To induce complete global ischemia, decapitated heads were incubated for 4 min at room temperature (∼25°C). For immunoblotting, a crude whole-brain membrane fraction was prepared from these animal brains, and proteins were analyzed on immunoblotting as described previously (Misonou et al., 2004). For immunohistochemical analyses, sagittal brain sections (each 40 μm thick) were prepared from perfusion-fixed animals (Rhodes et al., 2004). The sections were blocked with 10% goat serum and then stained with a rabbit anti-Kv2.1 antibody (Trimmer, 1991) using Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) as described previously (Rhodes et al., 2004). Fluorescent images were taken with a 24-bit color digital camera installed on a Zeiss (Oberkochen, Germany) Axiovert 200M microscope with a 63×, 1.3 numerical aperture lens and a grating imaging system, Apotome, using Axiovision software.
Materials. Unless stated otherwise, all reagents were obtained from Sigma (St. Louis, MO) or Roche (Indianapolis, IN).
Neuronal culture. Dissociated cultures of embryonic rat hippocampal neurons were prepared as previously described (Misonou et al., 2004).
Biochemical analysis of neuronal proteins. Samples for biochemical analyses were prepared from neuronal cultures as described previously (Misonou et al., 2004). Briefly, neurons were washed twice with HBSS and incubated with drugs as indicated in the figure legends. The cells were then washed twice with ice-cold Locke's solution (in mm: 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1 MgCl2, 5 glucose, and 5 HEPES, pH 7.4), harvested, and centrifuged at 12,000 × g for 30 min at 4°C. The pellets were extracted by adding sample buffer (2% SDS, 1 mm EDTA, 10% glycerol, 0.001% bromophenol blue, 5% β-mercaptoethanol, and 62.5 mm Tris-HCl, pH 6.8) and sonicating briefly. Rat brain membranes were prepared and treated with purified phosphatases as described previously (Misonou et al., 2004). Proteins were separated on 7.5% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-Kv2.1 mouse monoclonal antibody K89/41 (Antonucci et al., 2001) or anti-Kv4.2 mouse monoclonal antibody K57/1 (Rhodes et al., 2004). The blots were incubated with HRP-conjugated secondary antibodies (MP Biomedicals, Irvine, CA) followed by enhanced chemiluminescence reagent (PerkinElmer, Wellesley, MA). Immunoreactive bands were visualized by exposing the blot to x-ray film. Because the Kv2.1 population of relative electrophoretic mobility (Mr) ≈125 kDa represents the major constitutively phosphorylated form of neuronal Kv2.1 (Murakoshi et al., 1997), here we quantified the relative amounts of this species as a measure of Kv2.1 phosphorylation state (Misonou et al., 2004). Immunoreactive bands were quantified after scanning using NIH ImageJ software (http://rsb.info.nih.gov/ij/) with the gel plotting macro. Quantitation was performed on samples from at least three different cultures.
ATP assay. Neurons were incubated with 5 mm 2-deoxy-d-glucose and 5 mm sodium azide for 5-15 min to induce chemical ischemia (CI), washed twice with ice-cold Locke's solution, and harvested in lysis buffer (1% Triton X-100 and 10 mm Tris-HCl, pH 8.0). As a control, neurons were incubated without the CI reagents, but the reagents were added immediately before the wash step. The suspension was centrifuged at 16,000 × g for 10 min at 4°C to remove insoluble materials. The resultant lysates were analyzed for ATP levels by using an ATP assay kit based on a luciferin-luciferase method.
Immunofluorescence staining of neurons. Neurons were incubated with drugs as indicated in the figure legends and then fixed and stained with K89/41 and anti-microtubule-associated protein-2 (MAP-2) mouse monoclonal antibodies and Alexa-conjugated isotype-specific secondary antibodies (Invitrogen) as described previously (Misonou et al., 2004). Fluorescent images were taken with a 24-bit color digital camera installed on a Zeiss Axioskop 2 microscope with a 63×, 1.3 numerical aperture lens, using Axiovision software. Images were transferred to Adobe PhotoShop software (Adobe Systems, San Jose, CA) as JPEG files. Quantitative analyses of Kv2.1 clustering were done using raw images. The percentage of cells exhibiting diffuse Kv2.1 staining was determined by collecting images from 100 cells from each of two independent experiments (Lim et al., 2000).
Ca2+ imaging. Neurons were washed twice with HBSS supplemented with 10 mm HEPES, pH 7.4, and then loaded with 5 μm Fluo-4-AM (Invitrogen) for 5 min at room temperature (23-25°C). After washing out excess dye, cells were further incubated for 15 min to allow de-esterification of AM ester. Neurons were imaged at 1 Hz using confocal laser scanning microscopy (Zeiss LSM510) with argon laser (488 nm) anda40×, 1.3 numerical aperture lens. Fluorescence intensity was measured on the NIH ImageJ software (http://rsb.info.nih.gov/ij/). Values were expressed as ratio of fluorescence intensity (F) against the basal intensity (F0), ΔF/F0. For the analyses of spontaneous Ca2+ transients, frequencies of Ca2+ transients with ΔF/F0 > 0.2 were measured and expressed as percentages of the values in control. Alternatively, total Ca2+ loads by Ca2+ transients were measured by integrating traces of Ca2+ signal for 2 min.
Electrophysiology. IK current recordings were made with the whole-cell voltage-clamp configuration. Patch pipettes were pulled from borosilicate glass tubing, heat-polished at the tip to give a resistance of 1-2 MΩ when filled with the pipette solution (in mm: 140 KCl, 5 EGTA, 2 MgCl2, 10 glucose, and 10 HEPES, pH 7.3). Currents were recorded with a patch-clamp amplifier (EPC-10; HEKA Elektronik, Lambrecht/Pfalz, Germany), sampled at 10 kHz, and filtered at 2 kHz by a digital Bessel filter. All currents were capacitance and series resistance compensated and leak subtracted by standard P/n procedure. External solution contained 0.1 μm tetrodotoxin (TTX), 140 mm NaCl, 5 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm glucose, and 10 mm HEPES, pH 7.3. CI (5 mm 2-deoxy-d-glucose and 5 mm sodium azide) was applied to neurons for 15 min using a polytetrafluorethylene-glass multiple-barrel perfusion system. Currents were recorded from neurons before and after induction of CI. All recordings were performed at room temperature (23-25°C). The membrane potential was held at -100 mV and depolarized from the holding potential of -100 mV to voltages between -90 and +80 mV in 10 mV increments for 200 ms. Before the start of the test depolarization, a single prepulse to -10 mV was given for 30 ms to inactivate majority of the transient outward K+ currents. Peak outward sustained K+ currents (I) were measured 100 ms into the pulse at each depolarizing potential and were converted to conductance (G) using the equation, G = I/(V - Ek). The Nernst K+ equilibrium potential EK was calculated as -84 mV. The normalized conductances were plotted against the test potential (V) and fitted to a single Boltzmann equation G = Gmax/(1 + exp[-(V - V1/2)/k]), where Gmax is the maximum conductance, V1/2 is the test potential at which the IK channels have a half-maximal conductance, and k represents the slope of the activation curve.
Viability assay. Viability of neurons was assessed using the LIVE/DEAD assay kit (Invitrogen). Cells were incubated with CI reagents in HBSS for 10 min, washed twice with HBSS, and incubated with HBSS in the presence or absence of 5 mm tetraethylammonium (TEA) or 10 nm iberiotoxin for 1 h. Cells were then washed twice with HBSS and brought back to the normal culture condition. Cell viability was assessed after 24 h incubation. Numbers of live cells (calcein-positive cells, in green) and dead cells (ethidium homodimer-1 positive cells, in red) were counted in five areas (∼7.5 mm2 each) imaged with a 2.5× objective in three different cultures, and the viability was expressed as percentage viable cells, relative to control (untreated) cultures. Minor populations of nonneuronal cells in these relatively pure hippocampal cultures were excluded from the count.
Statistical analyses. All data are presented as the mean ± SEM. Normal data were compared by a two-sided t test. A t value of p < 0.05 was considered to be statistically significant.
CO2 anesthesia rapidly induces dephosphorylation of rat brain Kv2.1
The delayed rectifier Kv2.1 channel α subunit is a six-transmembrane segment polypeptide with large cytoplasmic N and C termini that contain more than 60 candidate phosphorylation sites. On SDS-polyacrylamide gels, the major forms of Kv2.1 in rat brain and cultured embryonic rat hippocampal neurons exhibit substantially higher Mr (≈125 and ≈115 kDa) than predicted from the deduced Kv2.1 primary sequence (95.3 kDa). In vitro alkaline phosphatase (AP) treatment of extracts prepared from either brain or cultured neurons shifts the Mr of Kv2.1 to ≈100 kDa (Misonou et al., 2004) (Fig. 1B), suggesting that Kv2.1 is constitutively and highly phosphorylated in both brain and cultured hippocampal neurons.
In response to revised institutional guidelines for animal care, we began to use CO2 gas to deeply anesthetize animals before they were decapitated. However, we found that Kv2.1 in samples prepared from the brains of CO2-anesthetized rats exhibited an altered Mr (≈100 kDa) that was similar to that of Kv2.1 in AP-treated samples, and distinct from the mobility of Kv2.1 (≈125 kDa) in previous samples prepared from animals decapitated without CO2 anesthesia (Fig. 1A). When we examined the effects of various forms of anesthesia on the phosphorylation state of Kv2.1 in vivo, we found that CO2 was unique in causing this significant shift in the Mr of Kv2.1 (Fig. 1A). The differences in Mr of Kv2.1 in samples from control and CO2-treated animals were eliminated by AP treatment (Fig. 1B,C), showing that they were attributable solely to changes in Kv2.1 phosphorylation state. The Mr of two related rat brain Kv channel α subunits, Kv4.2 (Fig. 1B) and Kv1.1 (data not shown), which also undergo significant Mr shifts during in vitro AP treatment, were not altered by the brief CO2 anesthesia. We have shown previously that kainate-induced seizures elicit dephosphorylation of Kv2.1 in vivo (Misonou et al., 2004). That the CO2-induced dephosphorylation of Kv2.1 shown here was independent of CO2-induced seizure activity was shown by the fact that the levels of phosphorylated Kv2.1 were not significantly different (p = 0.09; n = 3) between rats subjected to CO2 inhalation in the absence or presence of deep pentobarbital anesthesia (Fig. 1D).
CO2 anesthesia dramatically changes the subcellular localization of neuronal Kv2.1 in vivo
Kv2.1 is abundantly expressed in virtually all mammalian brain neurons (Trimmer and Rhodes, 2004) and is present in large somatodendritic clusters (Trimmer, 1991) that disperse in response to kainate-induced seizures (Misonou et al., 2004). To address whether CO2 anesthesia altered Kv2.1 clustering, pentobarbital-anesthetized rats were subjected to brief (2 min) CO2 inhalation and then rapidly perfused with paraformaldehyde fixative to minimize postmortem ischemic effects. In brains of control (pentobarbital anesthetized) rats, immunofluorescence staining for Kv2.1 was present as distinct surface clusters on somata and proximal dendrites of neurons in hippocampus and subiculum (Fig. 2), as well as cerebral cortex, thalamus, hypothalamus, and striatum (data not shown). In brains from CO2-treated animals, these high-density clusters of Kv2.1 were dispersed such that Kv2.1 staining was now uniform on the surface membrane in virtually all neurons in the hippocampal formation (Fig. 2) and in all other brain regions examined (data not shown). In contrast, the localization of other neuronal K+ channels (Kv4.2, Kv1.4, and BK/Slo) was not significantly altered in the brains of the CO2-treated animals (data not shown). Optical sectioning (Lanni and Wilson, 2000) of the Kv2.1-stained brain sections revealed that Kv2.1 remained in the neuronal membrane after CO2 treatment (supplemental Fig. 1A, available at www.jneurosci.org as supplemental material), suggesting that the change in Kv2.1 localization in the brains of CO2-treated animals was attributable to a lateral dispersion in the plasma membrane. CO2 anesthesia also led to disruption of the unusually large (2-5 μm diameter) Kv2.1 clusters (supplemental Fig. 1B, available at www.jneurosci.org as supplemental material) associated with muscarinic synapses in motor neurons (Muennich and Fyffe, 2003). These results demonstrate that brief CO2 inhalation in both conscious and unconscious rats rapidly changes the phosphorylation state and localization of Kv2.1 in vivo and, in some cells, their association with specialized synapses. As such, previous results obtained from CO2-anesthesized animals may have yielded an underestimation of the full extent of Kv2.1 phosphorylation and clustering in brain neurons. Moreover, these dynamic changes in Kv2.1 localization in vivo could impact its role in regulating somatic and dendritic excitability.
Ischemia induces Kv2.1 dephosphorylation in culture and in vivo
To begin to investigate the neuronal signaling mechanisms underlying the dramatic effects of CO2 inhalation in vivo, we examined the effects of CO2 exposure and acidified culture medium, mimicking tissue acidosis, on Kv2.1 in cultured hippocampal neurons. We found that the phosphorylation state of Kv2.1 was not altered during exposure of neurons to pH 6.6 medium or elevated CO2 for 20 min (Fig. 3A) mixed in ambient air to avoid excessive anoxia. However, inhibition of oxidative ATP synthesis, such as occurs in conditions of hypoxia/ischemia, by exposing cultured neurons to CI (5 mm sodium azide and 5 mm 2-deoxy-d-glucose) (Swanson and Benington, 1996) for 10 min induced Kv2.1 dephosphorylation (Fig. 3B). The intracellular ATP levels in cultured neurons were decreased to 17.1 ± 2.0% of control levels by 5 min of CI and to 8.8 ± 2.0% by 15 min of CI, consistent with a previous report (Bernstein and Bamburg, 2003). Although blockade of voltage-dependent Na+ channels with TTX partially but not significantly (p = 0.08) inhibited dephosphorylation of Kv2.1 induced by 10 min of CI (Fig. 3B), CI for 15 min even in the presence of 1 μm TTX yielded full induction of Kv2.1 dephosphorylation (Fig. 3C), suggesting that the CI effects occur essentially independent of membrane depolarization. The CI-induced Kv2.1 dephosphorylation was completely reversible during CI washout (supplemental Fig. 2A, available at www.jneurosci.org as supplemental material). Thus, as shown previously for glutamate-induced Kv2.1 dephosphorylation (Misonou et al., 2004), neurons can reestablish the normal Kv2.1 phosphorylation state after this brief ischemic insult.
The efficacy of CI but not media acidosis nor CO2 itself on Kv2.1 dephosphorylation in cultured neurons led us to reinvestigate the basis for CO2-induced Kv2.1 dephosphorylation in vivo. Brains from pentobarbital-anesthetized rats were subjected to complete global ischemia for 4 min after decapitation (Namba et al., 2002), which induced Kv2.1 dephosphorylation in vivo (Fig. 3D). Overdose itself with pentobarbital (150 mg/kg) over a 4 min period did not elicit Kv2.1 dephosphorylation (Fig. 3D), indicating that Kv2.1 dephosphorylation is not rapid postmortem changes. Together, these results suggest that the CO2 effects in vivo are primarily attributable to hypoxia but not hypercapnia and that metabolic inhibition by hypoxia/ischemia in vivo and by CI in vitro induces rapid dephosphorylation of Kv2.1.
Chemical ischemia modulates the phosphorylation state and localization of Kv2.1 through calcineurin activation
We next began to investigate the signaling pathway underlying the CI-induced dephosphorylation of Kv2.1. We found that, unlike the effects of glutamate (Misonou et al., 2004), the CI effects occurred in the absence of extracellular Ca2+ (Fig. 4A). Antagonists for ionotropic glutamate receptors (GluRs) (AP-5 and CNQX; GluR blockers) that predictably blocked the effects of glutamate (Misonou et al., 2004) did not affect induction by CI (Fig. 4B), showing that the CI effects were not mediated by secondary and incidental glutamate release. This conclusion was further supported by the result that blockade of exocytosis by botulinum neurotoxin type B also did not affect CI-induced Kv2.1 dephosphorylation, although the toxin effectively reduced the levels of vesicle-associated membrane protein-2 (data not shown).
The effects of glutamate on Kv2.1 phosphorylation in cultured hippocampal neurons are mediated by calcineurin (Misonou et al., 2004). Interestingly, the CI-induced effects on Kv2.1 dephosphorylation were also significantly (p = 0.003) blocked by the calcineurin inhibitors cyclosporin A (Fig. 4C) and FK520 (5 μm), a more widely available FK506 analog (supplemental Fig. 2B, available at www.jneurosci.org as supplemental material). These results suggest a CI-induced signaling pathway distinct in its upstream events from that induced by glutamate but similar in its convergence on calcineurin-dependent Kv2.1 dephosphorylation.
Given that CO2 inhalation in vivo and glutamate stimulation in vitro (Misonou et al., 2004) lead to both Kv2.1 dephosphorylation and dispersion of Kv2.1 clusters, we next examined the effects of CI on Kv2.1 localization in cultured neurons. Cultured hippocampal pyramidal neurons have distinct Kv2.1 clusters on their somatodendritic membrane (Fig. 4D) similar to those seen on pyramidal neurons in vivo (Fig. 2). CI caused dispersion of somatodendritic Kv2.1 clusters in cultured neurons (Fig. 4E), similar to the effects of glutamate stimulation (Misonou et al., 2004). As such, the majority of neurons treated with CI (74.7 ± 3.6%) showed diffuse somatodendritic Kv2.1 staining compared with a small minority of cells (13.3 ± 1.7%) in control cultures. Note that these cultured neurons have an additional pool of Kv2.1 on the axon initial segment, a culture artifact (Lim et al., 2000) not seen in brain neurons (Fig. 2), and this pool of Kv2.1 was not affected by glutamate or CI treatment. Importantly, the effects of CI on Kv2.1 localization were significantly inhibited by cyclosporin A (31.4 ± 2.2% cells showed a diffuse Kv2.1 pattern; p < 0.001 compared with CI-treated cells in the absence of cyclosporin A) (Fig. 4 F), supporting a role for calcineurin in the CI-induced effects on Kv2.1 phosphorylation and localization.
Chemical ischemia increases [Ca2+]i by inducing Ca2+ release from intracellular stores
Given the lack of a requirement for extracellular Ca2+ in the CI-induced effects on Kv2.1 and that increased [Ca2+]i is necessary and sufficient to induce the calcineurin-dependent dephosphorylation of Kv2.1 during glutamate stimulation (Misonou et al., 2004), we next investigated whether the CI effects were mediated by Ca2+ release from intracellular stores. Ca2+ imaging of cultured hippocampal neurons was performed using Fluo-4 and confocal laser scanning microscopy, focusing on changes in Fluo-4 fluorescence intensity on the soma and proximal dendrites in which Kv2.1 is localized. Hippocampal neurons exhibited spontaneous [Ca2+]i transients whose frequency and amplitude were suppressed by 1 μm tetrodotoxin (data not shown). CI was then induced, leading to a gradual increase in Fluo-4 signal intensity over 5 min (Fig. 5 A, B), as reported previously (Chow and Haddad, 1998). As shown in Figure 5B, CI induced a large and sustained increase in [Ca2+]i in the absence of extracellular Ca2+. This CI-induced increase in [Ca2+]i exhibited slower kinetics than that induced by glutamate stimulation in the presence of extracellular Ca2+, as reported for cultured cortical neurons (Chow and Haddad, 1998). However, the CI-induced increases in [Ca2+]i reached levels comparable with those seen during glutamate stimulation within 5 min of CI induction (Fig. 5B).
To pharmacologically investigate the source of intracellular Ca2+ release, we examined the effects of thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum (ER) Ca2+-ATPase caffeine to induce Ca2+ release from ryanodine-sensitive Ca2+ stores, and carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), which induces Ca2+ release from mitochondria by eliminating the H+ gradient (Yang et al., 2003). Neither thapsigargin nor caffeine induced significant Kv2.1 dephosphorylation (data not shown). However, treatment with FCCP alone resulted in an increase in [Ca2+]i similar to that obtained with CI (Fig. 5C), as well as dephosphorylation of Kv2.1 (supplemental Fig. 2C, available at www.jneurosci.org as supplemental material) in the absence of extracellular Ca2+.Importantly, FCCP treatment after induction of CI yielded no additional increases in [Ca2+]i (Fig. 5C), suggesting that the CI- and FCCP-induced Ca2+ release was from the same intracellular pool (i.e., mitochondria). Together, these data suggest that CI induces Ca2+ release from intracellular stores, presumably from mitochondria, and that the resultant increase in [Ca2+]i is sufficient to induce calcineurin-dependent dephosphorylation of Kv2.1.
Chemical ischemia alters the voltage-dependent activation of neuronal IK and neuronal activity
Whole-cell patch-clamp recording from cultured hippocampal neurons revealed that CI led to large hyperpolarizing shifts (control, V1/2 of +16.6 ± 0.4 mV; CI, V1/2 of -4.3 ± 0.4 mV; p < 0.001 vs control; n = 5) in the voltage dependence of activation of IK, without any significant change in the slope of the activation curve (Fig. 6A,B). These CI-induced changes are comparable with those induced by glutamate stimulation (Misonou et al., 2004). As a result of the CI-induced change in voltage-dependent activation, increased IK amplitude was observed at each test potential (Fig. 6A,B). These CI-induced shifts in voltage-dependent activation of IK were blocked by pretreatment with FK520 (V1/2 of +13.1 ± 0.5 mV) (Fig. 6B), suggesting that the calcineurin-dependent dephosphorylation of Kv2.1 was responsible for the altered biophysical properties of IK. Previous studies have shown that the bulk (≥80%) of IK in hippocampal and cortical pyramidal neurons is contributed by Kv2.1 (Murakoshi and Trimmer, 1999; Du et al., 2000; Pal et al., 2003). We found that the broad-spectrum K+ channel blocker TEA when used at 5 mm [the Ki for TEA block of Kv2.1 is 1-5 mm based on studies of recombinant rat brain Kv2.1 in heterologous cells (Shi et al., 1994; Immke et al., 1999)] effectively blocked (90.0 ± 0.2% inhibition) IK in control neurons. These data suggest that TEA effectively blocks the major Kv2.1 component of IK. Interestingly, a similar level of TEA inhibition was obtained for the increased IK in neurons after induction of CI (92.0 ± 1.4% inhibition), suggesting that the bulk of the enhanced IK in ischemic neurons can be attributed to Kv2.1. A mixture of iberiotoxin (IbTx, 10 nm), a specific blocker of large-conductance Ca2+-activated BK K+ channels, and apamin (500 nm), a specific blocker of small-conductance Ca2+-activated SK channels, yielded minimal block (12.0 ± 13.3% inhibition) of the total outward K+ current in these neurons.
Because such large changes in the activation properties of neuronal IK would be predicted to dramatically alter neuronal excitability (Surmeier and Foehring, 2004), we next examined whether CI changes the electrical activity of neurons, using spontaneous [Ca2+]i transients as an indirect readout of activity. As shown in Figure 6C, control hippocampal neurons exhibited regular spontaneous [Ca2+]i transients. Neurons subjected to CI (10 min) and washout (10 min, which restores resting [Ca2+]i) (Fig. 5B) showed a significantly (≈50%; p = 0.005 compared with control) depressed frequency of spontaneous [Ca2+]i transients of ΔF/F0 > 0.2 (Fig. 6C,D). The overall Ca2+ load (obtained by integrating changes in spontaneous [Ca2+]i transients over a 2 min period) was also significantly reduced (supplemental Fig. 3A, available at www.jneurosci.org as supplemental material). Furthermore, FK520 pretreatment reversed this suppression (p = 0.04) (supplemental Fig. 3A, available at www.jneurosci.org as supplemental material), indicating that the postischemic suppression of neuronal excitability results from calcineurin-dependent events.
A neuroprotective mechanism conferred by TEA-sensitive and iberiotoxin-insensitive K+ channels
To further understand the role of IK/Kv2.1 modulation in the suppression of neuronal activity, we examined whether TEA could reverse the suppression of neuronal excitability induced by CI. As shown in Figure 7, neurons incubated with 5 mm TEA (which blocks >90% of IK in these cultured hippocampal neurons; see above) showed initial burst activity followed by recovery of relatively normal spontaneous Ca2+ transients. However, TEA treatment of neurons that had been subjected to CI for 10 min elicited Ca2+ overload (Fig. 7) (supplemental Fig. 3B,C, available at www.jneurosci.org as supplemental material). In contrast to the dramatic effects of TEA, 10 nm IbTx did not have any noticeable effect on CI-induced [Ca2+]i, at least within the 4 min window of imaging (data not shown). These results show that, after brief ischemic insults, neurons exhibit hyperexcitability that can be suppressed by TEA-sensitive IbTx-insensitive IK currents.
To examine whether IK-mediated suppression of excitability would affect neuronal viability after brief ischemic insults, we assessed the neuronal damage induced by CI in the presence of TEA. Neurons were subjected to CI for 10 min, washed, and then incubated with or without TEA for 1 h. Cell viability was quantified after 24 h incubation under normal culture conditions. As shown in Figure 8, brief CI in itself did not affect neuronal viability, suggesting that neurons have protective mechanisms against this extent of ischemic insult. However, brief exposure of cells to 5 mm TEA just after CI resulted in significant neuronal damage, whereas TEA treatment in itself had little effect on cell viability (Fig. 8B). IbTx (10 nm) did not affect neuronal viability in the presence (Fig. 8B) or absence (data not shown) of ischemic insult. These results suggest that TEA-sensitive and IbTx-insensitive IK channels, of which a large component is Kv2.1, confer protection against ischemic neuronal damage by suppressing neuronal hyperexcitability.
Together, these results suggest that hypoxia/ischemia in vivo and in vitro induces calcineurin-mediated Kv2.1 dephosphorylation that leads to dramatic changes in its localization and function. Such modulation of Kv2.1 represents a novel protective mechanism activated in response to brief ischemia insults.
The major findings of this study are that brief ischemic insults in vivo and in vitro induce dephosphorylation of Kv2.1, loss of Kv2.1 clustering, and a large hyperpolarizing shift in the voltage-dependent activation of Kv2.1/IK channels. The ischemia-induced modulation of Kv2.1 is distinct from that induced in central neurons by seizures in vivo and by glutamate stimulation in vitro (Misonou et al., 2004) in that it occurs in the absence of extracellular Ca2+ and is not dependent on ischemia-induced seizures in vivo or glutamate release in vitro (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). The effects of ischemia in vivo are also more widespread and are observed throughout the brain, whereas the effects induced by kainate-induced seizures are restricted to the limbic system (Misonou et al., 2004). Despite these distinctions, increased [Ca2+]i and calcineurin remain central to both responses. Moreover, both the glutamate- and ischemia-induced effects on Kv2.1 are reversible, in that removal of either stimulus leads to recovery of phosphorylated Kv2.1 and Kv2.1 surface clusters and restoration of the relatively depolarized voltage-dependent activation typical of control neurons. Although release of Ca2+ from intracellular stores, specifically mitochondria as opposed to glutamate-stimulated Ca2+ influx via ionotropic glutamate receptors, seems to be critical to the ischemia-induced modulation of Kv2.1, sustained increases in [Ca2+]i leading to activation of calcineurin remain key. As predicted for the effects of glutamate stimulation (Surmeier and Foehring, 2004; Misonou et al., 2005), brief ischemic insult and enhanced activation of Kv2.1 leads to a suppression of neuronal activity (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). The K+ channel blocker TEA eliminates CI-induced suppression of neuronal excitability, and TEA treatment after brief and otherwise nonlethal exposure to CI leads to cell death. These findings show that neurons can use different upstream signaling pathways to achieve reversible calcineurin-dependent regulation of Kv2.1 localization and function in response to both hyperexcitability and ischemia. These studies also provide evidence that the activity of Kv2.1, although classically defined as a strictly voltage-dependent delayed rectifier K+ channel, is also dependent on metabolic state and [Ca2+]i.
Potential roles for Kv2.1 modulation in brain ischemia
Neurons need protection against excessive hyperactivity, which could lead to pathological excitotoxic conditions. It is widely accepted that neurons have developed a diverse array of immediate and long-term homeostatic protective mechanisms that respond to abnormal hyperactivity. Hypoxia and ischemia cause membrane depolarization, and the generalized seizures that can be evoked by such metabolic stress can aggravate brain damage (Calabresi et al., 2003). However, different neurons have distinct immediate and long-term protective mechanisms “on demand” to suppress the hyperexcitability induced by brief hypoxia/ischemia. One immediate mechanism is the depression of neuronal excitability during hypoxia/ischemia-induced activation of neuronal KATP channels (Ballanyi, 2004), especially in neurons in anoxia-tolerant brain regions such as substantia nigra pars reticulata (Yamada et al., 2001), dorsal vagal neurons, and cerebellar Purkinje cells (Ballanyi, 2004). However, many brain neurons (e.g., hippocampal pyramidal neurons) express lower levels of functional KATP channels (Zawar and Neumcke, 2000; Griesemer et al., 2002), such that brief hypoxia and ischemia in hippocampal slices induces immediate seizure-like hyperactivity followed by a delayed and prolonged suppression of neuronal activity (Kawasaki et al., 1990; Yamamoto et al., 1997). In cultured hippocampal neurons, the suppressed neuronal activity in response to hypoxia/ischemia is attributable to an increase in overall K+ current. Previous studies have attributed this increase to large-conductance Ca2+-activated BK channels, based on the fact that the increases in K+ current are dependent on increased [Ca2+]i (Yamamoto et al., 1997; Erdemli et al., 1998; Tanabe et al., 1999), although more recent studies used more definitive pharmacological blockade (Runden-Pran et al., 2002). That ischemia also modulates Kv2.1 in a Ca2+-dependent manner, through the activation of the protein phosphatase calcineurin, suggests that Kv2.1/IK may also contribute to ischemia-induced increases in neuronal K+ current. Moreover, the bulk of whole-cell IK in hippocampal neurons under both control and CI conditions, as well as the CI-induced suppression of neuronal excitability, are blocked by TEA doses that yield effective blockade of Kv2.1 and BK channels but not by the specific BK channel blocker IbTx. Previous studies showed that knockdown of Kv2.1/IK in cultured hippocampal slices by antisense oligonucleotides leads to neuronal hyperexcitability, but only under periods of high-frequency stimulation (Du et al., 2000). Conversely, when Kv2.1/IK is potentiated by arachidonic acid, neurons are protected from 4-aminopyridine-induced hyperexcitability (Colbert and Pan, 1999). These findings suggest that Kv2.1 can indeed act as a potent suppressor of neuronal hyperexcitability. Together with the results presented here, we suggest that, like KATP and BK channels, Kv2.1 can also couple metabolic state and [Ca2+]i to membrane excitability under periods of hypoxia/ischemia. However, for Kv2.1, such coupling occurs via a more indirect and slower mechanism than for the intrinsically ATP- and Ca2+-sensitive K+ channels, acting through dynamic and reversible calcineurin-dependent changes in phosphorylation state of the channel protein (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). Given the extremely high level of Kv2.1 expression in the somata and proximal dendrites of hippocampal (and virtually all other central) neurons, this Kv channel may represent a key but previously unrecognized contributor to the metabolic state- and Ca2+-dependent regulation of neuronal excitability.
Short- versus long-term effects of Kv2.1 modulation
Given the dynamic regulation of Kv2.1 function by reversible changes in phosphorylation state, it is not surprising that the role of Kv2.1/IK in neuroprotection is complex. Increases in delayed rectifier K+ channel activity in response to severe hypoxia and ischemia have been observed previously in CA1 hippocampal neurons (Chi and Xu, 2000, 2001; Zou et al., 2005), in large aspiny neostriatal neurons (Deng et al., 2005), in substantia nigra neurons (Jiang and Haddad, 1993), and in dorsal vagal motor neurons (Cowan and Martin, 1992). In cultured cortical neurons, increased TEA-sensitive IK in response to proapoptotic treatments (long-term serum deprivation or staurosporine treatment) is critical to the induction of apoptosis, such that TEA is antiapoptotic (Yu et al., 1997, 1999b; Wang et al., 2000; Wei et al., 2003). Models linking IK-mediated K+ efflux to altered [K+]i homeostasis, cell shrinkage, and proapoptotic cytochrome c release from mitochondria, as well as to direct K+ regulation of caspase, have been proposed (Yu et al., 1999b; Wei et al., 2003; Hribar et al., 2004). Knockdown experiments in cultured cortical neurons revealed that Kv2.1 is the specific TEA-sensitive Kv channel underlying the proapoptotic IK activity (Pal et al., 2003). However, we and others (Katsuki et al., 2005) found that TEA treatment exacerbated, not attenuated, neuronal injury induced by brief ischemia. Differences in the severity of insults and the modes of cell death involved, as well as the specific experimental system under study, may impact the effects of IK/Kv2.1 modulation on neuronal survival. The bulk of the previous studies (Yu et al., 1997, 1999a,b; Pal et al., 2003; Wei et al., 2003) showing proapoptotic roles for IK/Kv2.1 exposed neurons to relatively long (several hours to days) insults to cause extensive and sustained K+ efflux, cell shrinkage, and neuronal apoptosis. In our in vitro ischemia model, acute and brief CI (∼15 min) in itself was not sufficient to kill neurons but together with TEA treatment induced hyperexcitability and cell death, presumably by an excitotoxic non-apoptotic pathway. Because brain ischemia can kill neurons via both apoptosis and necrosis (Zhang et al., 2004), the complex neuroprotective and/or proapoptotic roles proposed for IK/Kv2.1 may also reflect Kv2.1 modulation in these different contexts. Our results are also consistent with a previous study that TEA does not protect against necrotic neuronal death induced by brief ischemic insult but provides neuroprotection against apoptotic cell death induced by longer, more intense insults (Wei et al., 2003). In addition, the timing of TEA addition relative to that of the ischemic insult can dramatically impact the neuroprotective effects of TEA in vivo (Huang et al., 2001).
We suggest that Kv2.1 might have a dual role in response to neuronal insults: an immediate protective role against acute hyperexcitability induced by seizures (Misonou et al., 2004) and brief (∼2-15 min) hypoxia attributable to calcineurin-dependent dephosphorylation, and altered localization and function, leading to homeostatic suppression of neuronal hyperexcitability. Removal of the ischemic (or excitotoxic) insult leads to a dynamic reversal of the calcineurin-dependent dephosphorylation and a recovery of the normal Kv2.1 phosphorylation state, clustering, and voltage-dependent activation. However, sustaining Kv2.1 in the dephosphorylated state under conditions of prolonged insult may lead to excessive K+ efflux, cell shrinkage, and apoptotic cell death. Thus, the role of Kv2.1 in neuronal survival in response to ischemic and other insults may be biphasic (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). Brief and reversible activation of Kv2.1 by dephosphorylation may be advantageous attributable to neuroprotective suppression of neuronal excitability, but sustained activation may eventually lead to deleterious effects via lowered [K+]i.
Possible mechanisms of ischemia-induced modulation of Kv2.1
Electron microscopic analysis has shown that plasma membrane Kv2.1 clusters on the somata and proximal dendrites of pyramidal neurons lie over subsurface cisternae (Du et al., 1998), intracellular ER-derived membranes rich in inositol triphosphate receptors and ryanodine receptors, and associated with high concentrations of mitochondria (Berridge, 1998; Paspalas and Goldman-Rakic, 2004). We found that, in cultured hippocampal neurons plasma membrane, Kv2.1 clusters overlap clusters of ryanodine receptors and the luminal Ca2+-binding protein calsequestrin (Antonucci et al., 2001; Misonou et al., 2005), suggesting a specialized neuronal Ca2+ signaling domain that may also contain a high density of calcineurin (Cameron et al., 1995). That FCCP, but not thapsigargin and caffeine, mimicked the effects of CI on modulation of Kv2.1, and that FCCP and CI treatments together yielded no additional increase in [Ca2+]i than achieved with CI alone suggest that mitochondria, especially ER-associated mitochondria (Berridge, 1998; Paspalas and Goldman-Rakic, 2004), are good candidates for the source of rapid intracellular Ca2+ release induced by ischemia. A number of recent studies have revealed the critical role for neuronal mitochondria in regulating Ca2+-dependent signaling events (Yang et al., 2003; Li et al., 2004). Moreover, mitochondrial function can be disturbed under such pathological conditions, for example, Ca2+ is released from mitochondria through the 2Na+/Ca2+ exchanger in hippocampal neurons during oxygen-glucose deprivation (Zhang and Lipton, 1999). Ca2+ stores in the cistern itself are an additional source of CI-induced Ca2+ release, as occurs in ischemic spinal cord neurons (Ouardouz et al., 2003). The localization of Kv2.1 at sites close to subsurface cisterns and associated mitochondria may allow for more efficient functional coupling of Ca2+ released from these intracellular stores to modulation of plasma membrane Kv2.1 channels. Moreover, such assemblies may allow for local “cluster-by-cluster” or quantal regulation of discrete pools of Kv2.1 in a manner distinct from the more graded response expected for a more uniformly distributed channel population. Dispersion of these Kv2.1 clusters during calcineurin-mediated dephosphorylation could provide a mechanism for more efficient reversibility and recovery, by removing Kv2.1 from this region of local Ca2+ and calcineurin signaling. The coupling of neuronal metabolism and [Ca2+]i to dynamic changes in Kv2.1 localization and function via regulation of the Kv2.1 phosphorylation state provides an effective and reversible mechanism for homeostatic regulation of central neuron excitability under conditions of ischemic and excitotoxic stress.
This research was supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke Grant NS42225 (J.S.T.), The Epilepsy Foundation (H.M.), and American Heart Association Grant 0565091Y (H.M.). Confocal imaging was conducted in a facility constructed with support from National Center for Research Resources, National Institutes of Health Research Facilities Improvement Program Grant Number C06 RR-12088-01. We thank Drs. R. Adamson and E. Yamoah for help with Ca2+ imaging and Drs. K. Rhodes, J. Antognini, M. O'Donnell, and D. E. Logothetis for helpful discussion.
Correspondence should be addressed to Hiroaki Misonou, Department of Pharmacology, School of Medicine, One Shields Avenue, University of California, Davis, Davis, CA 95616. E-mail:.
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