Lack of the Kir4.1 channel subunit abolishes K+ buffering properties of astrocytes in the ventral respiratory group: impact on extracellular K+ regulation

Ongoing rhythmic neuronal activity in the ventral respiratory group (VRG) of the brain stem results in periodic changes of extracellular K+. To estimate the involvement of the weakly inwardly rectifying K+ channel Kir4.1 (KCNJ10) in extracellular K+ clearance, we examined its functional expression in astrocytes of the respiratory network. Kir4.1 was expressed in astroglial cells of the VRG, predominantly in fine astrocytic processes surrounding capillaries and in close proximity to VRG neurons. Kir4.1 expression was up-regulated during early postnatal development. The physiological role of astrocytic Kir4.1 was studied using mice with a null mutation in the Kir4.1 channel gene that were interbred with transgenic mice expressing the enhanced green fluorescent protein in their astrocytes. The membrane potential was depolarized in astrocytes of Kir4.1-/- mice, and Ba2+-sensitive inward K+ currents were diminished. Brain slices from Kir4.1-/- mice, containing the pre-B?tzinger complex, which generates a respiratory rhythm, did not show any obvious differences in rhythmic bursting activity compared with wild-type controls, indicating that the lack of Kir4.1 channels alone does not impair respiratory network activity. Extracellular K+ measurements revealed that Kir4.1 channels contribute to extracellular K+ regulation. Kir4.1 channels reduce baseline K+ levels, and they compensate for the K+ undershoot. Our data indicate that Kir4.1 channels 1) are expressed in perineuronal processes of astrocytes, 2) constitute the major part of the astrocytic Kir conductance, and 3) contribute to regulation of extracellular K+ in the respiratory network.     

Differential regulation of Kir4.1 and Kir2.1 expression in the ischemic rat retina

Ischemia-reperfusion of the rat retina causes gliosis of Müller cells that is associated with a decrease of their K+ conductance. By using quantitative PCR and immunohistochemical staining of retinal slices, we investigated the effect of transient ischemia-reperfusion on retinal expression of two inward-rectifying K+ (Kir) channels, Kir4.1 and Kir2.1. In control retinas, Müller cells prominently expressed both Kir4.1 and Kir2.1 proteins. At 7 days after reperfusion, the expression of Kir4.1 protein was strongly downregulated, while the Kir2.1 protein expression remained unaltered. The expression of Kir4.1 mRNA was reduced by 55% after ischemia while the expression of Kir2.1 mRNA was not altered. The data suggest that the glial expression of distinct Kir channels is differentially regulated after retinal ischemia, with deletarious consequences for K+ ion and water homeostasis.     

Role and mechanisms of regulation of the basolateral Kir4.1/Kir5.1K+ channels in the distal tubules

Epithelial K⁺ channels are essential for maintaining electrolyte and fluid homeostasis in the kidney. It is recognized that basolateral inward‐rectifying K⁺ (K_ir) channels play an important role in the control of resting membrane potential and transepithelial voltage, thereby modulating water and electrolyte transport in the distal part of nephron and collecting duct. Monomeric K_ir4.1 (encoded by Kcnj10 gene) and heteromeric K_ir4.1/K_ir5.1 (K_ir4.1 together with K_ir5.1 (Kcnj16)) channels are abundantly expressed at the basolateral membranes of the distal convoluted tubule and the cortical collecting duct cells. Loss‐of‐function mutations in KCNJ10 cause EAST/SeSAME tubulopathy in humans associated with salt wasting, hypomagnesaemia, metabolic alkalosis and hypokalaemia. In contrast, mice lacking K_ir5.1 have severe renal phenotype that, apart from hypokalaemia, is the opposite of the phenotype seen in EAST/SeSAME syndrome. Experimental advances using genetic animal models provided critical insights into the physiological role of these channels in electrolyte homeostasis and the control of kidney function. Here, we discuss current knowledge about K⁺ channels at the basolateral membrane of the distal tubules with specific focus on the homomeric K_ir4.1 and heteromeric K_ir4.1/K_ir5.1 channels. Recently identified molecular mechanisms regulating expression and activity of these channels, such as cell acidification, dopamine, insulin and insulin‐like growth factor‐1, Src family protein tyrosine kinases, as well as the role of these channels in NCC‐mediated transport in the distal convoluted tubules, are also described.     

Differential distribution of spinal cord field potentials

Summary Electrical stimulation of the sural, superficial peroneal and plantar nerves in anesthetized cats produces a sequence of potentials in the spinal cord lumbosacral enlargement. The distributions of the spinal cord dorsum negative intermediary potential (N1 wave) and of the associated field potential recorded in depth from the spinal gray matter were mapped. The N1 wave produced by the sural nerve was largest at the junction of the S1 and L7 segments, whereas that evoked by the other two nerves was maximum in L6 and L7. The field potentials recorded in depth also showed a differential distribution. The maximum negativity during phase 2, corresponding to the N1 cord dorsum potential, was found to lie laterally in the dorsal horn when the sural nerve was stimulated, but medially when the plantar nerve was activated. The superficial peroneal nerve produced its largest negative field potential in the central region of the dorsal horn. The negative field potentials from the sural and superficial peroneal nerves were not as well separated spatially from each other as they were from the potential evoked by the plantar nerve.     

内流性钾通道2.1蛋白在大鼠脊髓和小脑的分布

目的：内流性钾通道(Kir)2．1蛋白在大鼠脊髓和小脑的分布。方法：免疫荧光组织化学和免疫荧光双标记。结果：在脊髓灰质前角的运动神经元和白质的星形胶质细胞呈现Kir2．1免疫反应活性强阳性。少数少突胶质细胞呈现Kir2．1和GS共存。在小脑蒲肯野氏细胞及纤维呈Kir2．1免疫活性强阳性。双标记显示近蒲肯野氏细胞旁的少突胶质细胞的Kir2．1蛋白和GS蛋白共存。结论：脊髓和小脑灰质和白质中的Kir2．1表达不一，这种分布可能与保持细胞外的K 平衡、促进K 的内流维持细胞兴奋性有重要关系。     

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.     

Kappa opioid receptors in rat spinal cord: sex-linked distribution differences

Activation of kappa opioid receptors (KORs) in the spinal cord can diminish nociception. Humans and rodents show sex differences in the analgesia produced by KOR agonists, and female rats show fluctuations in KOR density and sensitivity across the estrous cycle. However, it is unclear whether there are sex differences in the amount and/or distribution of spinal KORs. In the present study, immunocytochemically labeled KORs were examined in laminae I and II of the lumbosacral spinal dorsal horn of male and normally cycling female Sprague-Dawley rats. The basic pattern of KOR labeling was determined in both sexes using qualitative electron microscopy (EM), and sex-linked differences in the density and subcellular distribution of KOR immunoreactivity were determined with quantitative EM and light microscopy. KOR labeling was visualized with immunoperoxidase for optimally sensitive detection, or with immunogold for precise subcellular localization. By EM, the general pattern of KOR immunoreactivity was similar in males and females. KOR immunoreactivity was common in dendrites, axons, and axon terminals, and was in a few glia and neuronal somata. Most KOR-immunoreactive (-ir) axons were fine-diameter and unmyelinated. Most KOR-ir terminals were small or medium-sized, and a minority formed asymmetric or symmetric synapses with unlabeled dendrites. KOR immunoreactivity was associated both with the plasma membrane and with cytoplasmic organelles, notably including dense core vesicles in terminals. Light microscopic densitometry revealed that KOR immunoreactivity was significantly denser in estrus and proestrus females than in males. By EM, the distribution of KOR-immunogold labeling within axon terminals differed, with a greater proportion of cytoplasmic KOR labeling in estrus females compared with males. In contrast, the abundance and types of KOR-immunoperoxidase-labeled profiles did not show sex-linked differences. We conclude that in both sexes, KORs are positioned to influence both pre- and postsynaptic neurotransmission and are present in morphologically heterogeneous neuron populations. These findings are consistent with complex consequences of KOR activation in the spinal cord. In addition, the presence of increased KOR density and proportionally elevated intracellular KORs in proestrus/estrus females suggests a basis for sex-linked differences in KOR-mediated antinociception.     

Heterooligomeric assembly of inward-rectifier K+ channels from subunits of different subfamilies: Kir2.1 (IRK1) and Kir4.1 (BIR10)

 Activities of strong inward-rectifier K⁺ channels composed of K_ir2.1(84 M), K_ir2.1(84T) and K_ir4.1 subunits and weak inward-rectifier K⁺ channels composed of K_ir4.1(E158N) subunits were measured from giant inside-out patches of Xenopus laevis oocytes. The conductance/voltage (g/V) relationship for block by intracellular spermine (SPM) was biphasic for both K_ir2.1 channel types while it was monophasic for both K_ir4.1 channel types. The release of blocking Mg²⁺ ions was slow for K_ir2.1(84T) but virtually instantaneous for K_ir2.1(84M) and both K_ir4.1 channel types. Coexpression of K_ir2.1(84T) and K_ir4.1(E158N) resulted in heterooligomeric channels which were strongly rectifying, with a g/V relationship for SPM-evoked block that was significantly different from that of either parental homooligomeric channel type. Block by intracellular Mg²⁺ was markedly stronger than that for K_ir4.1(E158N) channels, while release of the block was almost instantaneous, similar to that for K_ir4.1(E158N) channels. This suggests preferential formation of a particular heterooligomer such as was recently proposed for subunits within the K_ir3.0 family. Received: 10 June 1996 / Received after revision and accepted: 26 July 1996     

Characterization of Na+-activated K+ currents in larval lamprey spinal cord neurons

Potassium channels play an important role in controlling neuronal firing and synaptic interactions. Na(+)-activated K(+) (K(Na)) channels have been shown to exist in neurons in different regions of the CNS, but their physiological function has been difficult to assess. In this study, we have examined if neurons in the spinal cord possess K(Na) currents. We used whole cell recordings from isolated spinal cord neurons in lamprey. These neurons display two different K(Na) currents. The first was transient and activated by the Na(+) influx during the action potentials, and it was abolished when Na(+) channels were blocked by tetrodotoxin. The second K(Na) current was sustained and persisted in tetrodotoxin. Both K(Na) currents were abolished when Na(+) was substituted with choline or N-methyl-D-glucamine, indicating that they are indeed dependent on Na(+) influx into neurons. When Na(+) was substituted with Li(+), the amplitude of the inward current was unchanged, whereas the transient K(Na) current was reduced but not abolished. This suggests that the transient K(Na) current is partially activated by Li(+). These two K(Na) currents have different roles in controlling the action potential waveform. The transient K(Na) appears to act as a negative feedback mechanism sensing the Na(+) influx underlying the action potential and may thus be critical for setting the amplitude and duration of the action potential. The sustained K(Na) current has a slow kinetic of activation and may underlie the slow Ca(2+)-independent afterhyperpolarization mediated by repetitive firing in lamprey spinal cord neurons.     

Kir4.1 expression by astrocytes and oligodendrocytes in CNS white matter: a developmental study in the rat optic nerve

Deletion studies in transgenic mice indicate that the potassium inward rectifying channel Kir4.1 is crucial for oligodendrocyte differentiation and has a special role in regulation of extracellular potassium (K(+)), a major function of astrocytes. However, there are conflicting reports on whether Kir4.1 is expressed by white matter astrocytes and oligodendrocytes, raising doubts over its functions. Here, we have examined Kir4.1 expression in astrocytes and oligodendrocytes of the rat optic nerve, a typical central nervous system white matter tract. Single and double immunofluorescence labelling was performed on frozen sections from optic nerves aged postnatal day (P)5, 10, 15, 20 and adult, using anti-Kir4.1 antibodies and the glia-specific antibodies glial fibrillary acidic protein (GFAP, astrocytes), carbonic anhydrase II (CAII, oligodendrocyte somata and processes) and myelin basic protein (MBP, oligodendrocyte myelin sheaths). The results demonstrate Kir4.1 expression in rows of glial cells as early as P5, and this pattern persisted throughout development and into adulthood, consistent with early expression of Kir4.1 on developing oligodendrocytes. Clear co-expression of Kir4.1 and GFAP is first evident at P10 and increases to adult levels by P15 and P20, which correlates with the development of K(+) regulation between P15 and P20. Astrocyte expression of Kir4.1 is localized to perivascular end-feet and fine processes within the fascicles of myelinated axons, consistent with a role in K(+) spatial buffering between nodes of Ranvier and blood vessels. By contrast, Kir4.1 is concentrated in the cell bodies of oligodendrocytes, and there is no apparent co-expression with MBP(+) myelin sheaths, suggesting oligodendroglial Kir4.1 channels are not involved in K(+) regulation. The results support roles for Kir4.1 in both oligodendrocyte differentiation and K(+) regulation by astrocytes.     

GFP imaging of live astrocytes: regional differences in the effects of ischaemia upon astrocytes

The basic division between white matter 'fibrous' astrocytes and grey matter 'protoplasmic' astrocytes is well established in terms of their morphological differences. The availability of transgenic animals with green fluorescent protein (GFP) expression restricted to specific glial cell types now provides an approach for looking at changes in cell number and morphology in the two astrocyte types in whole mount preparations. This is an important goal, as the ease of generating astrocyte cultures has led to a proliferation of studies that have examined ischaemic effects on astrocytes in vitro. This has in turn engendered a belief that astrocytes have an extraordinary resistance to ischaemic injury, a belief that runs counter to almost all the data available from in vivo and whole-mount preparations. One possible source of this confusion is the reactive changes that occur in astrocytes following injury, which include an increase in cell number that may obscure early astrocyte cell death and which has been reported to initiate within hours of an ischaemic event. However, we show here that neither white matter nor grey matter GFP(+) astrocytes exhibit any feature of reactive astrocytosis within a 180-min period of reperfusion following modelled ischaemia in neonatal whole-mount preparations. We also show that white matter astrocytes are much more sensitive to ischaemia-reperfusion injury than are grey matter astrocytes, a feature that may have high significance for developmental disorders of white matter tracts such as cerebral palsy.     

内向矫正性钾通道Kir4.1和Kir7.1在大鼠睫状体的分布

目的:在蛋白水平探讨Kir4.1和Kir7.1在大鼠睫状体的分布情况。方法:用间接免疫荧光组织化学和免疫荧光双标记在冰冻切片上检测Kir4.1和Kir7.1在大鼠睫状体的分布和定位。结果:Kir4.1蛋白分布于睫状体的色素上皮细胞和无色素上皮细胞的胞质和胞膜,内层的无色素上皮细胞免疫活性较强,免疫双标显示Kir4.1蛋白与谷氨酰胺合成酶蛋白重叠分布,Kir4.1蛋白免疫活性在睫状体的扁平部和睫状突上无明显差异。Kir7.1蛋白分布于睫状体的无色素上皮细胞和睫状肌,与特异性标记睫状上皮的Ezrin蛋白双标显示Kir7.1分布在无色素上皮的基底面,其免疫活性在睫状突强表达,在睫状体扁平部弱表达。结论:Kir4.1和Kir7.1均分布于大鼠睫状体的无色素上皮的基底面,可能与房水的生成有重要关系。睫状肌的Kir7.1分布,与调节睫状肌的舒缩、参与房水的排出密切相关。     

大鼠脊髓星形胶质细胞的分离与纯化

目的:纯化培养和鉴定新生大鼠的脊髓星形胶质细胞,建立稳定的星形胶质细胞培养体系,为研究创伤后慢性神经病理性痛的脊髓机制奠定基础。方法:根据成纤维细胞与星形胶质细胞生长倍增时间的差异,用阿糖胞苷(Ara-C)抑制成纤维细胞生长分裂的方法获取星形胶质细胞,用胶质原纤维酸性蛋白(GFAP)免疫荧光法对其鉴定。同时与差速贴壁培养法进行对比。结果:用Ara-C处理后,可获得较高纯度的星形胶质细胞,经鉴定其纯度可达90%以上,而对照组几乎全部为成纤维细胞。结论:Ara-C处理能彻底清除成纤维细胞,从而保持星形胶质细胞的正常生长繁殖,达到纯化星形胶质细胞的目的。     

Differential effect of alpha-syntrophin knockout on aquaporin-4 and Kir4.1 expression in retinal macroglial cells in mice

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.     

Differential distribution of 5-hydroxytryptamine and substance P in synaptosomal vesicles of rat ventral spinal cord

The subcellular distribution of 5-hydroxytryptamine (5-HT) and substance P (SP) was investigated in rat ventral spinal cord using differential and density gradient centrifugation. 5-HT was determined by high performance liquid chromatography (HPLC) and SP by radioimmunoassay (RIA). Both substances were found to be stored within synaptosomes at high densities. After partial lysis of the synaptosomes, 5-HT was also recovered in low density fractions, while substance P was found also in a fraction of intermediate density. The results indicate that within synaptosomes from ventral spinal cord, most of the 5-HT is stored in small synaptic vesicles, while SP is only stored in large synaptic vesicles. A minor part of the 5-HT may in addition also be stored in large vesicles, as previously suggested by ultrastructural studies.