Arachidonic acid activates an open rectifier potassium channel in cultured rat cortical astrocytes

A pathophysiological increase in free arachidonic acid (AA) is thought to regulate the channel-mediated astrocytic swelling occurring in several brain injuries. We report that in cultured rat type-1 cortical astrocytes, exposure to 10 microM AA activates an open rectifier K(+) channel, which exhibits many similarities with TREK/TRAAK members of the two-pore-domain K(+) channel family KCNK. Patch-clamp experiments showed that the current developed with a long latency and was preceded by a depression of the previously described outward rectifier K(+) conductance. Pharmacologic studies indicate that the K(+) open rectifier was differentially sensitive to classic K(+)-channel blockers (quinine, quinidine, tetraethylammonium, and barium) and was inhibited potently by gadolinium ions. The activation of this K(+) current occurred independently of the AA metabolism as pharmacologic inhibition of the lipoxygenase, cyclooxygenase, and cytochrome P450 epoxygenase signaling cascades did not alter the AA effect. Moreover, neither the neutralization of the NADPH-oxidase pathway nor scavenging intracellular free radicals modified the AA response. Finally, the AA-induced K(+) current was unaffected by protein kinase C inhibitors. The activation mechanism of the K(+) open rectifier was through an extracellular interaction of AA with the plasma membrane. RT-PCR analysis revealed that the AA-induced K(+) conductance was mediated likely by TREK-2 channels. Collectively, the results demonstrate that in cultured cortical astrocytes, pathological levels of AA directly activate an open rectifier K(+) channel, which may play a role in the control of K(+) homeostasis under pathophysiological conditions.     

Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1-deficient astrocytes

PURPOSE: Individuals with tuberous sclerosis complex (TSC) frequently have intractable epilepsy. To gain insights into mechanisms of epileptogenesis in TSC, we previously developed a mouse model of TSC with conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice). These mice develop progressive seizures, suggesting that glial dysfunction may be involved in epileptogenesis in TSC. Here, we investigated the hypothesis that impairment of potassium uptake through astrocyte inward rectifier potassium (Kir) channels may contribute to epileptogenesis in Tsc1(GFAP)CKO mice. METHODS: Kir channel function and expression were examined in cultured Tsc1-deficient astrocytes. Kir mRNA expression was analyzed in astrocytes microdissected from neocortical sections of Tsc1(GFAP)CKO mice. Physiological assays of astrocyte Kir currents and susceptibility to epileptiform activity induced by increased extracellular potassium were further studied in situ in hippocampal slices. RESULTS: Cultured Tsc1-deficient astrocytes exhibited reduced Kir currents and decreased expression of specific Kir channel protein subunits, Kir2.1 and Kir6.1. mRNA expression of the same Kir subunits also was reduced in astrocytes from neocortex of Tsc1(GFAP)CKO mice. By using pharmacologic modulators of signalling pathways implicated in TSC, we showed that the impairment in Kir channel function was not affected by rapamycin inhibition of the mTOR/S6K pathway, but was reversed by decreasing CDK2 activity with roscovitine or retinoic acid. Last, hippocampal slices from Tsc1(GFAP)CKO mice exhibited decreased astrocytic Kir currents, as well as increased susceptibility to potassium-induced epileptiform activity. CONCLUSIONS: Impaired extracellular potassium uptake by astrocytes through Kir channels may contribute to neuronal hyperexcitability and epileptogenesis in a mouse model of TSC.     

Downregulation of Kir4.1 inward rectifying potassium channel subunits by RNAi impairs potassium transfer and glutamate uptake by cultured cortical astrocytes

Glial cell-mediated potassium and glutamate homeostases play important roles in the regulation of neuronal excitability. Diminished potassium and glutamate buffering capabilities of astrocytes result in hyperexcitability of neurons and abnormal synaptic transmission. The role of the different K+ channels in maintaining the membrane potential and buffering capabilities of cortical astrocytes has not yet been definitively determined due to the lack of specific K+ channel blockers. The purpose of the present study was to assess the role of the inward-rectifying K+ channel subunit Kir4.1 on potassium fluxes, glutamate uptake and membrane potential in cultured rat cortical astrocytes using RNAi, whole-cell patch clamp and a colorimetric assay. The membrane potentials of control cortical astrocytes had a bimodal distribution with peaks at -68 and -41 mV. This distribution became unimodal after knockdown of Kir4.1, with the mean membrane potential being shifted in the depolarizing direction (peak at -45 mV). The ability of Kir4.1-suppressed cells to mediate transmembrane potassium flow, as measured by the current response to voltage ramps or sequential application of different extracellular [K+], was dramatically impaired. In addition, glutamate uptake was inhibited by knock-down of Kir4.1-containing channels by RNA interference as well as by blockade of Kir channels with barium (100 microM). Together, these data indicate that Kir4.1 channels are primarily responsible for significant hyperpolarization of cortical astrocytes and are likely to play a major role in potassium buffering. Significant inhibition of glutamate clearance in astrocytes with knock-down of Kir4.1 highlights the role of membrane hyperpolarization in this process.     

K(+) inward rectifier currents in reactive astrocytes from adult rat brain.

We characterized inward rectifier (K(IR)) currents in reactive astrocytes activated by CNS injury. We used primary cultures of reactive astrocytes obtained from gelatin sponge implants in adult rat brains, a system that yielded highly purified, homogeneous cultures with >95% of cells positive for GFAP, vimentin, and S-100beta. Ionic channels were studied in 1-21-day-old primary cultures using a nystatin-perforated patch clamp technique. Fast Na(+) currents were identified in <2% of cells. Most cells exhibited outward currents positive to -50 mV, with one component being sensitive to charybdotoxin, iberiotoxin, and tetraethylammonium chloride, and another component being sensitive to 4-aminopyridine. Two populations of cells were distinguished, based on presence or absence of Ba(2+)-sensitive K(IR) current negative to the K(+) reversal potential (E(k)), with >80% of cells expressing K(IR) currents. In contrast to previous reports on mammalian astrocytes, the current-voltage curve showed no appreciable current between E(k) and -50 mV, reflecting strong rectification by K(IR) channels. The magnitude of K(IR) current at -130 mV (I(-)(130)) did not change significantly during 21 days in culture (123 cells), suggesting constitutive expression of K(IR) channels. The fraction of K(IR)-negative cells was not affected by serum-starvation for 16-24 h. In cells with I(-)(130) >/= -30 pA, the membrane potential was invariably near E(k) and depolarized appreciably on addition of Ba(2+), but in cells with I(-)(130) < -30 pA, resting potentials ranged from -40 mV to -90 mV. We conclude that most adult reactive astrocytes constitutively express K(IR) channel(s) that exhibit strong rectification not previously observed in mammalian astrocytes.     

蛋白偶联内向整流钾通道的研究进展

G蛋白偶联内向整流钾通道(GIRK)是内向整流钾通道中的一个亚家族,由5个亚单元GIRK 1～5组成.它能调节心率及神经细胞兴奋性和静息电位的水平,起慢突触后抑制作用.本文主要阐述GIRK在基因结构、分布、调控和病理生理方面的研究进展.     

蛋白偶联内向整流钾通道的研究进展

G蛋白偶联内向整流钾通道(GIRK)是内向整流钾通道中的一个亚家族，由5个亚单元GIRK1～5组成。它能调节心率及神经细胞兴奋性和静息电位的水平，起慢突触后抑制作用。本文主要阐述GIRK在基因结构、分布、调控和病理生理方面的研究进展。     

Characterization of an inwardly rectifying chloride conductance expressed by cultured rat cortical astrocytes

The biophysical and pharmacological properties of the inwardly rectifying Cl⁻ conductance (I_Clh), expressed in rat type-1 neocortical cultured astrocytes upon a long-term treatment (1–3 weeks) with dibutyryl-cyclic-AMP (dBcAMP), were investigated with the whole-cell patch-clamp technique. Using intra- and extra-cellular solutions with symmetrical high Cl⁻ content and with the monovalent cations replaced with N-methyl-D-glucamine, time- and voltage-dependent Cl⁻ currents were elicited in response to hyperpolarizing voltage steps from a holding potential of 0 mV. The inward currents activated slowly and did not display any time-dependent inactivation. The rising phase of the current traces was best fitted with two exponential components whose time constants decreased with larger hyperpolarization. The steady-state activation of I_Clh was well described by a single Boltzmann function with a half-maximal activation potential at −62 mV and a slope of 19 mV that yields to an apparent gating charge of 1.3. The anion selectivity sequence was Cl⁻ = Br⁻ = I⁻ > F⁻ > cyclamate ≥ gluconate. External application of the putative Cl⁻ channel blockers 4,4 diisothiocyanatostilbene-2,2 disulphonic acid or 4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid did not affect I_Clh. By contrast, anthracene-9-carboxylic acid, as well as Cd²⁺ and Zn²⁺, inhibited, albeit with different potencies, the Cl⁻ current. Taken together, these results indicate that dBcAMP-treated cultured rat cortical astrocytes express a Cl⁻ inward rectifier, which exhibits similar but not identical features compared with those of the cloned and heterologously expressed hyperpolarization-activated Cl⁻ channel ClC-2. GLIA 21:217–227, 1997. © 1997 Wiley-Liss, Inc.     

The endocannabinoid anandamide inhibits potassium conductance in rat cortical astrocytes

Endocannabinoids are a family of endogenous signaling molecules that modulate neuronal excitability in the central nervous system (CNS) by interacting with cannabinoid (CB) receptors. In spite of the evidence that astroglial cells also possess CB receptors, there is no information on the role of endocannabinoids in regulating CNS function through the modulation of ion channel‐mediated homeostatic mechanisms in astroglial cells. We provide electrophysiological evidence that the two brain endocannabinoids anandamide (AEA) and 2‐arachidonylglycerol (2‐AG) markedly depress outward conductance mediated by delayed outward rectifier potassium current (IK_DR) in primary cultured rat cortical astrocytes. Pharmacological experiments suggest that the effect of AEA does not result from the activation of known CB receptors. Moreover, neither the production of AEA metabolites nor variations in free cytosolic calcium are involved in the negative modulation of IK_DR. We show that the action of AEA is mediated by its interaction with the extracellular leaflet of the plasma membrane. Similar experiments performed in situ in cortical slices indicate that AEA downregulates IK_DR in complex and passive astroglial cells. Moreover, IK_DR is also inhibited by AEA in NG2 glia. Collectively, these results support the notion that endocannabinoids may exert their modulation of CNS function via the regulation of homeostatic function of the astroglial syncytium mediated by ion channel activity. © 2008 Wiley‐Liss, Inc.     

Human angiotensinogen is highly expressed in astrocytes in human cortical grafts

Human fetal parietal cortical tissue was transplanted to cortical cavities in immunosuppressed rats. Protoplasmic astrocytes in the human cortical grafts highly expressed human angiotensinogen mRNA as identified with ³⁵S-labeled and digoxigenin-labeled riboprobes combined with immunohistochemistry for glial fibrillary acidic protein. Antibodies to human specific neurofilament protein 70 KD were used to characterize neurons in the graft and fiber outgrowth into the host brain. Immunohistochemistry revealed human angiotensinogen-like immunoreactivity in many small protoplasmic astrocytes and very few large neurons. These results demonstrate that human angiotensinogen mRNA and protein is synthesized in immature human glia. We assume that angiotensinogen is transformed into angiotensin peptides, which may participate in the regulation of growth processes. The results suggest that human angiotensinogen may play a role during human ombryogenesis. © 1994 Wiley-Liss, Inc.     

Single inward rectifier channels in horizontal cells

The ion channels responsible for inward rectification in horizontal cells were studied using the patch clamp technique applied to isolated cells from goldfish retina. Inward currents recorded from these cells were identified as due to the opening of inward rectifier channels based on their ion selectivity, channel gating behavior, and the effects of external blocking ions. The single channel conductance was 20 pS in 125 mM external K+. The null current potential shifted with changes in the K+ concentration as expected for a channel permeable to K+, and the channel appeared to have little permeability to Na+. The probability of a channel being in an open state increased as the membrane was hyperpolarized from the K+ equilibrium potential (0 to -10 mV) over potentials ranging to -80 mV, in the presence of external Na+. The closing rate was insensitive to membrane potential in the presence of external Na+. The opening rate of the channel increased as the membrane was hyperpolarized. The increase in the probability of a channel being open at negative potentials was therefore caused by the voltage sensitivity of the rate of channel opening.     

Sodium dependency of the inward potassium rectifier in horizontal cells isolated from the white bass retina

The ionic properties underlying the inwardly rectifying potassium current in cultured voltage-clamped white bass horizontal cells were studied. Anomalous rectification was apparent upon membrane hype rpolarization with a reversal potential depolarized from the predicted value of E_K In raised extracellular potassium, the current increased and the reversal potential shifted toward a more depolarized membrane potential. Solutions containing decreased sodium caused a rapid decrease in the inward rectifier current but only slightly affected the reversal potential. Extracellular cesium or barium caused a reversible voltage-dependent reduction of the inward current. We interpret these results to mean that the inward rectifying channel in white bass horizontal cells is mainly permeable to potassium ions, but is sodium dependent. It may shape the photoresponses of the horizontal cells and may contribute to a hyperpolarization activated conductance increase measured in situ.     

G‐protein‐coupled inward rectifier potassium channels involved in corticostriatal presynaptic modulation

Presynaptic modulation has been associated mainly with calcium channels but recent data suggests that inward rectifier potassium channels (K_IR) also play a role. In this work we set to characterize the role of presynaptic K_IR channels in corticostriatal synaptic transmission. We elicited synaptic potentials in striatum by stimulating cortical areas and then determined the synaptic responses of corticostriatal synapsis by using paired pulse ratio (PPR) in the presence and absence of several potassium channel blockers. Unspecific potassium channels blockers Ba²⁺ and Cs⁺ reduced the PPR, suggesting that these channels are presynaptically located. Further pharmacological characterization showed that application of tertiapin‐Q, a specific K_IR3 channel family blocker, also induced a reduction of PPR, suggesting that K_IR3 channels are present at corticostriatal terminals. In contrast, exposure to Lq2, a specific K_IR1.1 inward rectifier potassium channel, did not induce any change in PPR suggesting the absence of these channels in the presynaptic corticostriatal terminals. Our results indicate that K_IR3 channels are functionally expressed at the corticostriatal synapses, since blockage of these channels result in PPR decrease. Our results also help to explain how synaptic activity may become sensitive to extracellular signals mediated by G‐protein coupled receptors. A vast repertoire of receptors may influence neurotransmitter release in an indirect manner through regulation of K_IR3 channels. Synapse 69:446–452, 2015 . 2015 Wiley Periodicals, Inc.     

Voltage-dependent potassium currents in hypertrophied rat astrocytes after a cortical stab wound

Changes in the membrane properties of reactive astrocytes in gliotic cortex induced by a stab wound were studied in brain slices of 21-28-day-old rats, using the patch-clamp technique and were correlated with changes in resting extracellular K+ concentration ([K+]e) measured in vivo using K+-selective microelectrodes. Based on K+ current expression, three types of astrocytes were identified in gliotic cortex: A1 astrocytes expressing a time- and voltage-independent K+ current component and additional inwardly rectifying K+ currents (K(IR)); A2 astrocytes expressing a time- and voltage-independent K+ current component and additional delayed outwardly rectifying K+ currents (K(DR)); and complex astrocytes expressing K(DR), K(IR), and A-type K+ (K(A)) currents and Na+ currents (I(Na)). Nestin/bromodeoxyuridine (BrdU)-negative A1 astrocytes were found further than approximately 100 microm from the stab wound and showed an upregulation of K(IR) currents within the first day post-injury (PI), correlating with an increased resting [K+]e. Their number declined from 62% of total astrocytes in control rats to 41% in rats at 7 days PI. Nestin/BrdU-positive A2 astrocytes were found only within a distance of approximately 100 microm from the stab wound and, in comparison to those in control rats, showed an upregulation of K(DR) currents. Their number increased from 8% of the total number of astrocytes in control rats to 39% 7 days PI. Both A1 and A2 astrocytes showed hypertrophied processes and increased GFAP staining, but an examination of cell morphology revealed greater changes in the surface/volume ratio in A2 astrocytes than in A1 astrocytes. Complex astrocytes did not display a hypertophied morphology; K(IR) currents in these cells were upregulated within 1 day PI, while the K(DR), K(A), and I(Na) currents were increased only 6 h PI. We conclude that two electrophysiologically, immunohistochemically, and morphologically distinct types of hypertrophied astrocytes are present at the site of a stab wound, depending on the distance from the lesion, and may have different functions in ionic homeostasis and/or regeneration.     

The KCNQ5 potassium channel from mouse: a broadly expressed M-current like potassium channel modulated by zinc, pH, and volume changes

The KCNQ proteins compose a sub-group of the voltage-activated potassium channel family. The family consists of five members (KCNQ1 to 5--also named Kv7.1 to Kv7.5) encoded by single genes, which all give rise to proteins forming slowly activating potassium-selective ion channels. The physiological importance of the KCNQ channel family is emphasized by the fact that mutations in four of the five genes have been linked to human pathologies (KCNQ1 to 4). Here, we present the cloning and characterization of a novel KCNQ5 ortholog from mouse isolated by homology cloning from total mouse brain RNA (GenBank accession number: AY679158). The predicted protein is 95% identical to human KCNQ5. Upon expression in Xenopus oocytes, these proteins form voltage-dependent slowly activating channels with half-maximal activation at -21 mV. Our functional characterization revealed three novel modes of modulation: pH-dependent potentiation by Zn2+ (EC50 = 21.8 microM at pH 7.4), inhibition by acidification (IC50 = 0.75 microM; pKa = 6.1), and regulation by small changes in cell volume. Furthermore, the channels are activated by the anti-convulsant drug retigabine (EC50 = 2.0 microM) and inhibited by the M-current blockers linopiridine and XE-991. Finally, real-time RT-PCR was used to quantify the expression profile in a wide range of mouse tissues. These experiments revealed a relatively broad expression pattern in the nervous system but also expression in other tissues. Highest overall expression levels were observed in cortex and hippocampus. This study shows that murine KCNQ5 channels, in addition to sharing biophysical and pharmacological characteristics with the human ortholog, are tightly regulated by physiological stimuli such as changes in extracellular Zn2+, pH, and tonicity, thus adding to the complex regulation of these channels.     

Aberrant modulation of a delayed rectifier potassium channel by glutamate in Alzheimer's disease

In Alzheimer's disease (AD), potassium channel abnormalities have been reported in both neural and peripheral tissues. Herein, using whole-cell patch-clamp, we demonstrate an aberrant glutamate-dependent modulation of K_V1.3 channels in T lymphocytes of AD patients. Although intrinsic K_V1.3 properties in patients were similar to healthy individuals, glutamate (1–1000 μM) failed to yield the hyperpolarizing shift normally observed in K_V1.3 steady-state inactivation (? 4.4 ± 2.7 mV in AD vs. ? 14.3 ± 2.5 mV in controls, 10 μM glutamate), resulting in a 4-fold increase of resting channel activity. Specific agonist and antagonist data indicate that this abnormality is due to dysfunction of cognate group II mGluRs. Given that glutamate is present in plasma and that both mGluRs and K_V1.3 channels regulate T-lymphocyte responsiveness, our finding may account for the presence of immune-associated alterations in AD. Furthermore, if this aberration reflects a corresponding one in neural tissue, it could provide a potential target in AD pathogenesis.     

Relationship between inward rectifier potassium current impairment and brain injury after cerebral ischemia/reperfusion.

Functional alterations of barium-sensitive potassium inward rectifier (KIR) current, which is involved in the vasodilation of middle cerebral arteries (MCA) in rat brain, have been described during brain ischemia/reperfusion (I/R). The authors investigate the effects of I/R on KIR current recorded in isolated myocytes from MCA of control rats and from contralateral and ipsilateral MCA of ischemic rats by the whole-cell patch-clamp technique, and the relationship between its alteration and the severity of brain injury. The vascular smooth muscle cells exhibited similar morphologic features in all conditions, and the KIR was present in the three groups of myocytes, exhibiting a characteristic inward rectification and a normal external potassium dependence. The KIR density was significantly reduced in cell of MCA ipsilateral to occlusion with a maximum at -135 mV, whereas there was no difference between control and contralateral cells. This alteration in KIR density in occluded MCA was significantly correlated with severity of brain injury and brain edema. These results suggest that the alteration of KIR density in MCA myocytes after I/R and the consecutive impaired dilation of MCA may contribute to aggravation of the brain injury.     

Inward rectifier potassium channel Kir 2.3 is inhibited by internal sulfhydryl modification

Regions of the hippocampal inward rectifier potassium channel Kir 2.3 that contact the aqueous environment were investigated by identification of native cysteine residues that confer sulfhydryl reagent sensitivity to the channel conductance. Kir 2.3 currents were inhibited by N-ethylmaleimide (NEM), whereas currents of Kir 2.1 were unaffected. The reactive residues were identified as Kir 2.3 Cys28 and Cys50 using chimeric constructs and mutagenesis. These sites were not accessible to p-chloromercuriphenylsulfonate (pCMPS) applied extracellularly. However, both Cys28 and Cys50 were accessible to 2-(trimethylammoniumethyl) methanethiosulfonate (MTSET) applied to the intracellular surface of the membrane. These studies demonstrate that Cys28 and Cys50 lie in a cytoplasmic aqueous accessible region of the channel, and suggest that the channel N-terminus is a key constituent of the internal vestibule of the pore and/or modulates channel gating.