The Journal of Neuroscience, November 30, 2005, ():
Calcium- and Metabolic State-Dependent Modulation of the Voltage-Dependent Kv2.1 Channel Regulates Neuronal Excitability in Response to Ischemia
J. Neurosci. Misonou et al.
25: 11184
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Fig. 1 A, Surface localization of Kv2.1. Brain sections from control and CO2-treated rats were stained with anti-Kv2.1 antibody. The upper panels show projected images reconstituted from 40 optical sections (thickness 0.4 痠 per section) taken from subiculum. The lower panels show the side view of the cell bodies cut at the gray line in the top panels. Kv2.1 staining in pyramidal neurons from both control and CO2-treated rats shows a clear ring-shape pattern, indicating surface expression of Kv2.1. B, The effect of CO2 on Kv2.1 clustering in large neurons in the brainstem. The projected images were reconstituted from 40 optical sections (thickness 0.4 痠 per section) taken from the facial nucleus in the medulla oblongata. Scale bars, 10 痠.
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Supplemental Fig. 2 A, Recovery of the phosphorylated pool of Kv2.1 after chemical ischemia. Neurons were incubated without (C), or with CI (CI-wash) or 5 然 glutamate (Glu-wash) for 10 min, washed to remove the reagents, and then incubated in normal media for 0 to 2 h. B, Blockade of the effect of CI on Kv2.1 dephosphorylation by calcineurin inhibitors. Neurons were incubated without treatment (Control) or with 10 min CI following no pretreatment (Ischemia), or pretreatment with 20 然 cyclosporin A (Cyclosporin) or 5 然 FK520 for 10 min. C, Kv2.1 dephosphorylation induced by FCCP. Neurons were incubated without (control) or with CI (Ischemia) or with 1 然 FCCP (FCCP) for 15 min in the absence of extracellular Ca2+. Proteins were solubilized in SDS sample buffer, fractionated on SDS-PAGE, and analyzed for Kv2.1 by immunoblotting. Numbers to left denote mobility of molecular weight standards in kDa. RBM, rat brain membrane fraction. AP, alkaline phosphatase treated rat brain membrane fraction.
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Supplemental Fig. 3 A, Removal of the suppression of spontaneous Ca2+ transients after CI by FK520. Neurons were loaded with 5 然 Fluo-4, incubated with 5 然 FK520 for 10 min, subjected to CI for 10 min, and then washed. Relative Ca2+ load was assessed by integrating the traces of spontaneous Ca2+ transients for two minutes before (Control, open bars) and after CI (Ischemia, closed bars). *P<0.05. B, Ca2+ overload induced by tetraethylammonium (TEA) was assessed by averaging 10 data points before TEA addition, and 10 s, 80 s, and 180 s after TEA addition in control (open squares), TEA (open circles), TEA after ischemia (closed circles). C, Relative Ca2+ load induced by TEA was assessed by integrating the traces for two minutes after the addition of TEA. Ca2+ load is expressed relative to that of control cells.
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Supplemental Fig. 4 Schematic diagram of mechanisms inducing Kv2.1 modulation. We have shown that increased neuronal activity induced by glutamate or seizures elicits Ca2+ influx leading to calcineurin activation (Misonou et al., 2004). Hypoxia/ischemia induces Ca2+ release from intracellular Ca2+ stores, presumably from mitochondria, followed by calcineurin activation. These stimuli ultimately cause Kv2.1 modulation and result in suppression of intrinsic neuronal excitability that could serve as a reversible homeostatic mechanism to protect neurons from glutamate- and hypoxia/ischemia-induced hyperexcitability.
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