Calcium- and metabolic state-dependent modulation of the voltage-dependent Kv2.1 channel regulates neuronal excitability in response to ischemia

Abstract

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.

Figure 1.

CO₂ inhalation induces dephosphorylation of Kv2.1 in vivo. A, Effects of different anesthetics on the phosphorylation state of Kv2.1 in vivo. Animals were anesthetized by 2.5 min inhalation of CO₂, diethyl ether (Ether), isoflurane, or by intraperitoneal injection of 50 mg/kg pentobarbital, or 24 mg/kg ketamine and 3.2 mg/kg xylazine (Ketamine) and were then decapitated to prepare brain membrane fractions. Proteins were separated by SDS-PAGE and analyzed for Kv2.1 by immunoblotting. Numbers on the left indicate the mobility of molecular weight standards in kilodaltons. B, Crude membranes were prepared from brains of control and CO₂-treated animals and incubated without (control) or with 0.1 U/ml AP. Proteins were analyzed by immunoblotting for Kv2.1 and Kv4.2. C, Line scan analysis of immunoblots showing dramatic dephosphorylation of Kv2.1 in CO₂-treated animals. Bands of total and phosphorylated (M_r of 25 kDa) Kv2.1 were quantified. Numbers below the labeling indicate the amount of phosphorylated Kv2.1 as a percentage of total Kv2.1 level and are the mean ± SEM from three independent experiments. ^*p < 0.001 compared with control. AU, Arbitrary units. D, Effects of pentobarbital on CO₂-induced Kv2.1 dephosphorylation. Animals were injected with vehicle or 50 mg/kg sodium pentobarbital and then treated with CO₂ for 2 min. Crude brain membranes were prepared and analyzed by immunoblotting for Kv2.1.

Figure 2.

CO₂ dramatically changes localization of Kv2.1 in vivo. Rats were intraperitoneally injected with 50 mg/kg pentobarbital and exposed to air (control) or CO₂ for 2 min in a closed chamber. Brain sections from these control and CO₂-treated rats were stained with anti-Kv2.1 antibody. Images were taken by conventional fluorescence microscopy from the subiculum (S) and CA1 region of the hippocampus. Insets are higher-magnification views of Kv2.1 staining corresponding to the boxed areas in the main images. Scale bar, 10 μm.

Figure 3.

Ischemia induces dephosphorylation of Kv2.1 in vitro and in vivo. A, Cultured hippocampal neurons were incubated in buffer at pH 7.4 or 6.6 or treated in a chamber filled with CO₂ gas at 37°C for 20 min. Proteins were solubilized in SDS sample buffer, fractionated on SDS-PAGE, and analyzed for Kv2.1 by immunoblotting. B, Neurons were incubated with CI reagents (5 mm 2-deoxy-d-glucose and 5 mm sodium azide; Ischemia) in the presence or absence of 1 μm TTX for 10 min. Bands of total and phosphorylated (M_r of 125 kDa, indicated by an arrow) Kv2.1 were quantified. Values in boxes indicate the amount of phosphorylated Kv2.1 (M_r of 125 kDa) as a percentage of total Kv2.1 and are the mean values from five independent experiments (control, 62.0 ± 4.7%; ischemia, 21.0 ± 4.5%; ischemia plus TTX, 33.5 ± 0.5%). Values in parentheses denote the level of phosphorylated Kv2.1 shown as a percentage of the control level. Statistical significance between the control and experimental values are shown by asterisks (p < 0.01, n = 5). C, Neurons were incubated with CI reagents (Chemical ischemia) for 2, 5, 10, or 15 min in the presence of 1 μm tetrodotoxin. The levels of phosphorylated Kv2.1 were as follows: control, 100.0 ± 7.6%; 15 min of CI, 23.5 ± 3.1%. ^*p < 0.01 (n = 3). D, Animals were decapitated (Control) or injected with 150 mg/kg pentobarbital. To induce complete global ischemia, decapitated heads were incubated for 4 min at room temperature (Ischemia). Crude brain membranes were prepared from these animals, and proteins were analyzed by immunoblotting for Kv2.1. A fraction of crude membranes was incubated with 0.1 U/ml AP. The levels of phosphorylated Kv2.1 were as follows: control, 100.0 ± 3.3%; global ischemia, 55.4 ± 0.7%. Numbers to left or right denote mobility of molecular weight standards in kilodaltons.

Figure 4.

Chemical ischemia changes Kv2.1 phosphorylation state and localization by driving Ca²⁺ from the intracellular stores and activating calcineurin. A, Neurons were incubated with CI reagents for 2, 5, 10, and 15 min in the absence of extracellular Ca²⁺. Values in boxes indicate the level of phosphorylated Kv2.1 (marked by an arrow) shown as a percentage of the control level (control, 100 ± 6.4%; 15 min of CI, 34.1 ± 7.2%) (n = 3). B, Neurons were incubated with 20 μm glutamate or CI reagents (Ischemia) for 15 min, in the presence or absence of 100 μm AP-5 and 10 μm CNQX (GluR blockers) in the presence of 1 μm tetrodotoxin. The levels of phosphorylated Kv2.1 were as follows: control, 100 ± 6.8%; ischemia, 24.2 ± 2.2%; ischemia plus GluR blockers, 22.7 ± 2.4%; glutamate, 30.4 ± 4.6%; glutamate plus GluR blockers, 83.4 ± 1.7% (n = 3). RBM, Rat brain membrane fraction. C, Neurons were incubated with 20 μm glutamate or CI reagents for 15 min, in the presence or absence of 20 μm cyclosporin A in the presence of 1 μm tetrodotoxin. The levels of phosphorylated Kv2.1 were as follows: control, 100 ± 6.8%; glutamate, 28.4 ± 2.8%; glutamate plus cyclosporin A, 89.1 ± 5.5%; ischemia, 28.2 ± 5.4%; ischemia plus cyclosporin A, 86.5 ± 9.7% (n = 3). Proteins were solubilized in SDS sample buffer, fractionated on SDS-PAGE, and analyzed for Kv2.1 by immunoblotting. Statistical significance between control and experimental values (A, B) or between values in the presence and absence of cyclosporin A (C) are shown by asterisks (p < 0.01). Numbers to left or right denote mobility of molecular weight standards in kilodaltons. D-F, Neurons were incubated without (D) or with (E) CI reagents, or CI reagents and 20 μm cyclosporin A (F) for 15 min. Cells were fixed with 4% paraformaldehyde and stained with anti-Kv2.1 antibody (green) and anti-MAP-2 antibody (red).

Figure 5.

Chemical ischemia induces Ca²⁺ release from intracellular stores. A, Neurons were loaded with 5 μm Fluo-4 and incubated with CI reagents (5 mm 2-deoxy-d-glucose and 5 mm sodium azide) for 10 min in the presence of 1 μm tetrodotoxin. B, CI-induced increase of [Ca²⁺]_i in the absence of extracellular Ca²⁺. CI reagents or glutamate (10 μm) were added at the point indicated by an arrow. Neurons were incubated with glutamate in the presence of extracellular Ca²⁺. Thick yellow line represents the absence of recording during the washout period. C, Role of mitochondria as a source of released Ca²⁺. Neurons were incubated without (gray trace) or with (black trace) CI reagents for 10 min and then incubated with 1 μm FCCP for 5 min. Imaging rate, 1 Hz.

Figure 6.

Chemical ischemia alters the properties of I_K current and suppresses neuronal excitability. A, Representative I_K currents in a cultured hippocampal neuron recorded under whole-cell voltage clamp. 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. A 30 ms prepulse to -10 mV was given before each test depolarization to eliminate transient K⁺ current. B, The plot shows the conductance-voltage (G-V) relationship of peak I_K currents recorded from neurons before (open square), after (filled circle) CI, and after CI in the presence of 5 μm FK520 (filled square). C, Suppression of spontaneous Ca²⁺ transients after CI. Neurons were loaded with 5 μm Fluo-4. Representative spontaneous Ca²⁺ bursts in the soma before and after CI. Imaging rate, 1 Hz. D, Spontaneous Ca²⁺ bursts with ΔF/F₀ > 0.2 in the soma for 2 min were analyzed in cells before (Control) and after (Ischemia) the treatment with CI reagents. ^*p < 0.01 (n = 8).

Figure 7.

Tetraethylammonium induces Ca²⁺ overload in cultured neurons after ischemic insults. Neurons were loaded with 5 μm Fluo-4, incubated without or with CI reagents (5 mm 2-deoxy-d-glucose and 5 mm sodium azide) for 10 min, washed, and then incubated without or with 5 mm TEA. Representative traces of Ca²⁺ signals in cells treated with vehicle (Control), TEA-treated cells (TEA), and cells treated with TEA after ischemic insults (TEA after ischemia) are shown. The traces in the right bottom panel shows the means ± SEM of each data point in control (light gray), TEA (gray), and TEA after ischemia (black) from five independent cultures. The arrow indicates the time of TEA addition.

Figure 8.

Tetraethylammonium aggravates neuronal damage after ischemic insults. A, Neurons were incubated with CI reagents (5 mm 2-deoxy-d-glucose and 5 mm sodium azide) for 10 min, washed, and then incubated with 5 mm TEA for 1 h. After the incubation, cells were kept in the normal culture condition for 24 h and subjected to the cell viability assay as illustrated in the top schematic. All treatments were done in the absence of tetrodotoxin. Representative images taken with a 2.5× objective were shown in the bottom panels. Scale bar, 500 μm. B, The number of total and viable cells were counted (total of >200 cells were counted in each sample) and shown as percentages of viable cells in control. Iberiotoxin (10 nm) was added as described for TEA. Data are the means ± SEM (n = 5). ^*p < 0.01.