Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina.

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.     

Glial heterogeneity in expression of the inwardly rectifying K(+) channel, Kir4.1, in adult rat CNS

Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K(+) channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Müller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K(+) through this channel, whereas nonexpressors might use other mechanisms.     

Kir potassium channel subunit expression in retinal glial cells: implications for spatial potassium buffering

To understand the role of different K(+) channel subtypes in glial cell-mediated spatial buffering of extracellular K(+), immunohistochemical localization of inwardly rectifying K(+) channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward-rectifying K(+) channel subunit Kir4.1 and for the strongly inward-rectifying K(+) channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell-mediated transport of extracellular K(+) away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K(+) currents leave the glial cells and enter extracellular "sinks," whereas K(+) influxes from neuronal "sources" into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the "cables" for spatial buffering currents may prevent an unwarranted outward leak of K(+), and, thus, avoid disturbances of neuronal information processing.     

Expression of an inwardly rectifying K+ channel, Kir5.1, in specific types of fibrocytes in the cochlear lateral wall suggests its functional importance in the establishment of endocochlear potential

Cochlear endolymph contains 150 mm K+ and has a highly positive potential of approximately +80 mV. The specialized ionic composition and high potential in endolymph are essential for hearing and maintained by circulation of K+ from perilymph to endolymph through the cochlear lateral wall. Various types of K+ channel such as Kir4.1 and KCNQ1/KCNE1 are expressed in stria vascularis of the lateral wall and play essential roles in K+ circulation. In this study, we examined a distribution of another K+ channel, Kir5.1, and found it specifically expressed in the spiral ligament of the cochlear lateral wall. Specific immunoreactivity for Kir5.1 was detected in type II, IV and V fibrocytes of the ligament and spiral limbus, all of which are directly involved in K+ circulation. Kir5.1 was not found in either type I or III fibrocytes. Although Kir5.1 assembles with Kir4.1 to form a functional Kir channel in renal epithelia and retinal Müller cells, double-immunolabelling revealed that they were expressed in distinct regions in the cochlea lateral wall, i.e. Kir4.1 only in stria vascularis vs. Kir5.1 in spiral ligament. During development, the expression of Kir5.1 subunits started significantly later than Kir4.1 and was correlated with the 'rapid' phase of the elevation of endocochlear potential (EP). Kir5.1 and Kir4.1 channel-subunits may therefore play distinct functional roles in K+ circulation in the cochlear lateral wall.     

Evidence that compromised K+ spatial buffering contributes to the epileptogenic effect of mutations in the human Kir4.1 gene (KCNJ10)

Mutations in the human Kir4.1 potassium channel gene (KCNJ10) are associated with epilepsy. Using a mouse model with glia-specific deletion of Kcnj10, we have explored the mechanistic underpinning of the epilepsy phenotype. The gene deletion was shown to delay K(+) clearance after synaptic activation in stratum radiatum of hippocampal slices. The activity-dependent changes in extracellular space volume did not differ between Kcnj10 mutant and wild-type mice, indicating that the Kcnj10 gene product Kir4.1 mediates osmotically neutral K(+) clearance. Combined, our K(+) and extracellular volume recordings indicate that compromised K(+) spatial buffering in brain underlies the epilepsy phenotype associated with human KCNJ10 mutations.     

Kir subfamily in frog retina: specific spatial distribution of Kir 6.1 in glial (Müller) cells

We show by immunocytochemistry in frog retina that most members of the Kir subfamily are expressed in specific neuronal compartments. However, Kir 6.1, the pore-forming subunit of K(ATP) channels, is expressed exclusively in glial Müller cells. Müller cell endfeet display strong Kir 6.1 immunolabel throughout the retina, whereas the somata are labeled only in the retinal periphery. This spatial pattern is similar to that of Kir 4.1, of the ratio of inward to outward K+ currents, and of spermine/spermidine immunoreactivity. We suggest that the co-expression of Kir 4.1 and Kir 6.1 subunits may enable the cells to maintain their high K+ conductance and hyperpolarized membrane potentials both at high ATP levels (Kir 4.1) and during ATP deficiency (Kir 6.1).     

Identification of an inward rectifier potassium channel gene expressed in mouse cortical astrocytes

These experiments identify an inward rectifier K+ (Kir) channel expressed in mouse cortical and white matter astrocytes at the molecular level. Messenger RNA for one of the known Kir channel genes, Kir4.1, is present at much higher levels in cortical astrocytes in primary culture than the other known Kir family members. In culture, the level of Kir4.1 mRNA is lower in proliferating cells and in cells cultured for 16 h under hypoxic conditions, compared to confluent cells. Partial differentiation of the astrocytes with dibutyryl cAMP or by coculture with neurons has no effect on the Kir4.1 mRNA level. In situ hybridization experiments show that Kir4.1 mRNA is broadly distributed in the adult brain, including the neocortex, the stratum pyrimadale of the hippocampus, and the piriform cortex. Immunostaining confirms that the Kir4.1 protein is expressed by cultured astrocytes and also by cocultured cortical neurons. Astrocytes and neurons display a patchy pattern of immunostaining, raising the possibility that the channels sort themselves in clusters in the plasma membrane. Stellate cells in the neocortex and white matter are immunoreactive for Kir4.1, and double immunofluorescence experiments show colocalization of Kir4.1 and glial acidic fibrillary protein (GFAP) on stellate cells in the white matter. The cloned mouse Kir4.1 cDNA, when expressed heterologously in HEK cells, gives rise to inactivating Kir channels similar to those recorded from cultured astrocytes. These results indicate that the Kir4.1 gene product forms a Kir channel, or is a subunit of the channel, in mouse cortical astrocytes both in culture and in vivo.     

Functional expression of Kir4.1 channels in spinal cord astrocytes

Spinal cord astrocytes (SCA) have a high permeability to K+ and hence have hyperpolarized resting membrane potentials. The underlying K+ channels are believed to participate in the uptake of neuronally released K+. These K+ channels have been studied extensively with regard to their biophysics and pharmacology, but their molecular identity in spinal cord is currently unknown. Using a combination of approaches, we demonstrate that channels composed of the Kir4.1 subunit are responsible for mediating the resting K+ conductance in SCA. Biophysical analysis demonstrates astrocytic Kir currents as weakly rectifying, potentiated by increasing [K+]o, and inhibited by micromolar concentrations of Ba2+. These currents were insensitive to tolbutemide, a selective blocker of Kir6.x channels, and to tertiapin, a blocker for Kir1.1 and Kir3.1/3.4 channels. PCR and Western blot analysis show prominent expression of Kir4.1 in SCA, and immunocytochemistry shows localization Kir4.1 channels to the plasma membrane. Kir4.1 protein levels show a developmental upregulation in vivo that parallels an increase in currents recorded over the same time period. Kir4.1 is highly expressed throughout most areas of the gray matter in spinal cord in vivo and recordings from spinal cord slices show prominent Kir currents. Electrophysiological recordings comparing SCA of wild-type mice with those of homozygote Kir4.1 knockout mice confirm a complete and selective absence of Kir channels in the knockout mice, suggesting that Kir4.1 is the principle channel mediating the resting K+ conductance in SCA in vitro and in situ.     

Increased immunoreactivity of cdk5 activators in hippocampal sclerosis

Cyclin-dependent kinase 5 is important in several in-vitro neurodegeneration paradigms. Whether cyclin-dependent kinase 5 contributes to cell death in human neurodegenerative diseases remains uncertain, particularly because post-mortem delay and other extrinsic factors might influence cyclin-dependent kinase 5 activity. Here we demonstrate increased immunoreactivity for the activators of cyclin-dependent kinase 5 in post-mortem human hippocampi affected by the neurodegenerative condition hippocampal sclerosis, but not in histologically normal hippocampi. Moreover, in post-mortem brain tissue from patients with unilateral hippocampal sclerosis, increased immunoreactivity for cyclin-dependent kinase 5 activators was detected in the hippocampus with sclerosis, but not in the contralateral hippocampus, suggesting that extrinsic factors are unlikely to account for the differential staining observed. Our findings suggest that deregulation of cyclin-dependent kinase 5 might contribute to the pathogenesis of hippocampal sclerosis.     

Decreased expression of the glial water channel aquaporin-4 in the intrahippocampal kainic acid model of epileptogenesis

Recent evidence suggests that astrocytes may be a potential new target for the treatment of epilepsy. The glial water channel aquaporin-4 (AQP4) is expressed in astrocytes, and along with the inwardly-rectifying K(+) channel K(ir)4.1 is thought to underlie the reuptake of H(2)O and K(+) into glial cells during neural activity. Previous studies have demonstrated increased seizure duration and slowed potassium kinetics in AQP4(-/-) mice, and redistribution of AQP4 in hippocampal specimens from patients with chronic epilepsy. However, the regulation and role of AQP4 during epileptogenesis remain to be defined. In this study, we examined the expression of AQP4 and other glial molecules (GFAP, K(ir)4.1, glutamine synthetase) in the intrahippocampal kainic acid (KA) model of epilepsy and compared behavioral and histologic outcomes in wild-type mice vs. AQP4(-/-) mice. Marked and prolonged reduction in AQP4 immunoreactivity on both astrocytic fine processes and endfeet was observed following KA status epilepticus in multiple hippocampal layers. In addition, AQP4(-/-) mice had more spontaneous recurrent seizures than wild-type mice during the first week after KA SE as assessed by chronic video-EEG monitoring and blinded EEG analysis. While both genotypes exhibited similar reactive astrocytic changes, granule cell dispersion and CA1 pyramidal neuron loss, there were an increased number of fluorojade-positive cells early after KA SE in AQP4(-/-) mice. These results indicate a marked reduction of AQP4 following KA SE and suggest that dysregulation of water and potassium homeostasis occurs during early epileptogenesis. Restoration of astrocytic water and ion homeostasis may represent a novel therapeutic strategy.     

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.     

Correlation between calbindin expression in granule cells of the resected hippocampal dentate gyrus and verbal memory in temporal lobe epilepsy

Calbindin expression of granule cells of the dentate gyrus is decreased in temporal lobe epilepsy (TLE) regardless of its etiology. In this study, we examined the relation between reduction of calbindin immunoreactivity and the verbal and visuo-spatial memory function of patients with TLE of different etiologies. Significant linear correlation was shown between calbindin expression and short-term and long-term percent retention and retroactive interference in auditory verbal learning test (AVLT) of patients including those with hippocampal sclerosis. In addition, we found significant linear regression between calbindin expression and short-term and long-term percent retention of AVLT in patients whose epilepsy was caused by malformation of cortical development or tumor and when no hippocampal sclerosis and substantial neuronal loss were detected. Together with the role of calbindin in memory established in previous studies on calbindin knock-out mice, our results suggest that reduction of calbindin expression may contribute to memory impairments of patients with TLE, particularly, when neuronal loss is not significant.     

Role of Kir4.1 channels in growth control of glia

The inwardly rectifying potassium channel Kir4.1 is widely expressed by astrocytes throughout the brain. Kir4.1 channels are absent in immature, proliferating glial cells. The progressive expression of Kir4.1 correlates with astrocyte differentiation and is characterized by the establishment of a negative membrane potential (> -70 mV) and an exit from the cell cycle. Despite some correlative evidence, a mechanistic interdependence between Kir4.1 expression, membrane hyperpolarization, and control of cell proliferation has not been demonstrated. To address this question, we used astrocyte-derived tumors (glioma) that lack functional Kir4.1 channels, and generated two glioma cell lines that stably express either AcGFP-tagged Kir4.1 channels or AcGFP vectors only. Kir4.1 expression confers the same K+ conductance to glioma membranes and a similar responsiveness to changes in [K+]o that characterizes differentiated astrocytes. Kir4.1 expression was sufficient to move the resting potential of gliomas from -50 to -80 mV. Importantly, Kir4.1 expression impaired cell growth by shifting a significant number of cells from the G2/M phase into the quiescent G0/G1 stage of the cell cycle. Furthermore, these effects could be nullified entirely if Kir4.1 channels were either pharmacologically inhibited by 100 microM BaCl2 or if cells were chronically depolarized by 20 mM KCl to the membrane voltage of growth competent glioma cells. These studies therefore demonstrate directly that Kir4.1 causes a membrane hyperpolarization that is sufficient to account for the growth attenuation, which in turn induces cell maturation characterized by a shift of the cells from G2/M into G0/G1.     

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

A major role for Müller cells in the retina is to buffer changes in the extracellular K+ concentration ([K+]o) resulting from light-evoked neuronal activity. The primary K+ conductance in Müller cells is the inwardly rectifying K+ channel Kir4.1. Since this channel is constitutively active, K+ can enter or exit Müller cells depending on the state of the [K+]o. This process of [K+]o buffering by Müller cells ("K+ siphoning") is enhanced by the precise accumulation of these K+ channels at discrete subdomains of Müller cell membranes. Specifically, Kir4.1 is localized to the perivascular processes of Müller cells in animals with vascular retinas and to the endfeet of Müller cells in all species examined. The water channel aquaporin-4 (AQP4) also appears to be important for [K+]o buffering and is expressed in Müller cells in a very similar subcellular distribution pattern to that of Kir4.1. To gain a better understanding of how Müller cells selectively target K+ and water channels to discrete membrane subdomains, we addressed the question of whether Kir4.1 and AQP4 associate with the dystrophin-glycoprotein complex (DGC) in the mammalian retina. Immunoprecipitation (IP) experiments were utilized to show that Kir4.1 and AQP4 are associated with DGC proteins in rat retina. Furthermore, AQP4 was also shown to co-precipitate with Kir4.1, suggesting that both channels are tethered together by the DGC in Müller cells. This work further defines a subcellular localization mechanism in Müller cells that facilitates [K+]o buffering in the retina.     

Mitochondrial dysfunction disrupts trafficking of Kir4.1 in spiral ganglion satellite cells

The inward-rectifier K(+) channel Kir4.1 is responsible for maintaining cochlear homeostasis and restoring neural excitability. The large-conductance calcium-activated K(+) channel (BK(Ca)) plays a key role in phase locking signals in the mammalian inner ear. To evaluate the influence of mitochondrial dysfunction on the expression and subcellular localization of these channels, 3-nitropropionic acid (3-NP) was administered to rat round window membranes for 30 min. Auditory brainstem response was measured both before and 2 hr after 3-NP administration. Immunofluorescent confocal microscopy was used to measure the expression and subcellular localization of Kir4.1 and BK(Ca). Alexa Fluor 568-labeled bovine serum albumin (BSA) was applied to round window membranes as a tracer to explore the cochlear distribution of drug delivery and was detected in the lateral wall, spiral ganglion, cochlear nerve, and organ of Corti. Hearing loss of 23 (+/-4.4 SE) and 58 (+/-6.7 SE) dB developed in rats treated with 0.3 and 0.5 mol/liter of 3-NP, respectively. BK(Ca) was visualized in the cellular membrane and cytoplasm in the upper and middle region of inner hair cells, and it was not affected by 3-NP. Kir4.1 was detected in intermediate cells of the stria, Deiter's cells, and spiral ganglion satellite cells. Kir4.1 failed to reach the perineural cytoplasm of the satellite cells after 3-NP treatment. The results of this study suggest that mitochondrial dysfunction disrupts trafficking of Kir4.1 in spiral ganglion satellite cells.     

Aquaporin-4 independent Kir4.1 K+ channel function in brain glial cells

Functional interaction of glial water channel aquaporin-4 (AQP4) and inwardly rectifying K+ channel Kir4.1 has been suggested from their apparent colocalization and biochemical interaction, and from the slowed glial cell K+ uptake in AQP4-deficient brain. Here, we report multiple lines of evidence against functionally significant AQP4-Kir4.1 interactions. Whole-cell patch-clamp of freshly isolated glial cells from brains of wild-type and AQP4 null mice showed no significant differences in membrane potential, barium-sensitive Kir4.1 K+ current or current-voltage curves. Single-channel patch-clamp showed no differences in Kir4.1 unitary conductance, voltage-dependent open probability or current-voltage relationship. Also, Kir4.1 protein expression and distribution were similar in wild-type and AQP4 null mouse brain and in the freshly isolated glial cells. Functional inhibition of Kir4.1 by barium or RNAi knock-down in primary glial cell cultures from mouse brain did not significantly alter AQP4 water permeability, as assayed by calcein fluorescence quenching following osmotic challenge. These studies provide direct evidence against functionally significant AQP4-Kir4.1 interactions in mouse glial cells, indicating the need to identify new mechanism(s) to account for altered seizure dynamics and extracellular space K+ buffering in AQP4 deficiency.