Co-localisation of Kir4.1 and AQP4 in rat and human cochleae reveals a gap in water channel expression at the transduction sites of endocochlear K+ recycling routes

Sensory transduction in the cochlea depends on perilymphatic-endolymphatic potassium (K⁺) recycling. It has been suggested that the epithelial supporting cells (SCs) of the cochlear duct may form the intracellular K⁺ recycling pathway. Thus, they must be endowed with molecular mechanisms that facilitate K⁺ uptake and release, along with concomitant osmotically driven water movements. As yet, no molecules have been described that would allow for volume-equilibrated transepithelial K⁺ fluxes across the SCs. This study describes the subcellular co-localisation of the K_ir4.1?K⁺ channel (K_ir4.1) and the aquaporin-4 water channel (AQP4) in SCs, on the basis of immunohistochemical double-labelling experiments in rat and human cochleae. The results of this study reveal the expression of K_ir4.1 in the basal or basolateral membranes of the SCs in the sensory domain of the organ of Corti that are adjacent to hair cells and in the non-sensory domains of the inner and outer sulci that abut large extracellular fluid spaces. The SCs of the inner sulcus (interdental cells, inner sulcus cells) and the outer sulcus (Hensen??s cells, outer sulcus cells) display the co-localisation of K_ir4.1 and AQP4 expression. However, the SCs in the sensory domain of the organ of Corti reveal a gap in the expression of AQP4. The outer pillar cell is devoid of both K_ir4.1 and AQP4. The subcellular co-localisation of K_ir4.1 and AQP4 in the SCs of the cochlea described in this study resembles that of the astroglia of the central nervous system and the glial Mueller cells in the retina.     

Altered Kir and gap junction channels in temporal lobe epilepsy

Since astrocytes may sense and respond to neuronal activity these cells are now considered important players in brain signaling. Astrocytes form large gap junction coupled syncytia allowing them to clear the extracellular space from K⁺ and neurotransmitters accumulating during neuronal activity, and redistribute it to sites of lower extracellular concentrations. Increasing evidence suggests a crucial role for dysfunctional astrocytes in the etiology of epilepsy. Notably, alterations in expression, localization and function of astroglial K⁺ channels as well as impaired K⁺ buffering was observed in specimens from patients with pharmacoresistant temporal lobe epilepsy and in chronic epilepsy models. Altered astroglial gap junction coupling has also been reported in epileptic tissue which, however, seems to play a dual role: (i) junctional coupling counteracts hyperactivity by facilitating clearance of elevated extracellular K⁺ and glutamate while (ii) it also provides a pathway for energetic substrates and fuels neuronal activity. Dysfunctional astrocytes should be considered promising targets for new therapeutic strategies.     

Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Bötzinger complex

Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.     

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.     

Pannexin 1 activity in astroglia sets hippocampal neuronal network patterns

Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.

Astrocytes have mostly been shown to boost neuronal activity. This study reveals that activity-dependent activation of astroglial pannexin 1 channels inhibits hippocampal neuronal networks by decreasing neuronal excitability via purinergic signaling, uncovering a novel astroglial negative feedback loop mechanism.     

The role of extracellular potassium dynamics in the different stages of ictal bursting and spreading depression: A computational study

Experimental evidences point out the participation of nonsynaptic mechanisms (e.g., fluctuations in extracellular ions) in epileptiform bursting and spreading depression (SD). During these abnormal oscillatory patterns, it is observed an increase of extracellular potassium concentration [K⁺]_o and a decrease of extracellular calcium concentration [Ca²⁺]_o which raises the neuronal excitability. However, whether the high [K⁺]_o triggers and propagates these abnormal neuronal activities or plays a secondary role into this process is unclear. To better understand the influence of extracellular potassium dynamics in these oscillatory patterns, the experimental conditions of high [K⁺]_o and zero [Ca²⁺]_o were replicated in an extended Golomb model where we added important regulatory mechanisms of ion concentration as Na⁺-K⁺ pump, ion diffusion and glial buffering. Within these conditions, simulations of the cell model exhibit seizure-like discharges (ictal bursting). The SD was elicited by the interruption of the Na⁺−K⁺ pump activity, mimicking the effect of cellular hypoxia (an experimental protocol to elicit SD, the hypoxia-induced SD). We used the bifurcation theory and the fast-slow method to analyze the interference of K⁺ dynamics in the cellular excitability. This analysis indicates that the system loses its stability at a high [K⁺]_o, transiting to an elevated state of neuronal excitability. Effects of high [K⁺]_o are observed in different stages of ictal bursting and SD. In the initial stage, the increase of [K⁺]_o creates favorable conditions to trigger both oscillatory patterns. During the neuronal activity, a continuous growth of [K⁺]_o by outward K⁺ flow depresses K⁺ currents in a positive feedback way. At the last stage, due to the depression of K⁺ currents, the Na⁺-K⁺ pump is the main mechanism in the end of neuronal activity. Thus, this work suggests that [K⁺]_o dynamics may play a fundamental role in these abnormal oscillatory patterns.     

Tyrosine Phosphorylation of Kir3 following ��-Opioid Receptor Activation of p38 MAPK Causes Heterologous Desensitization

Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K_ir3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K_ir3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wild-type mice, but not in mice lacking the κ-opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K_ir3.1-ir. The U50,488-induced increase in pY12-K_ir3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K_ir3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K_ir3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K_ir3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K_ir3.1-ir; and the heterologous desensitization of K_ir3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K_ir3.1 and channel inhibition through a GRK3-, p38 MAPK- and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.     

Differential Expression of Potassium Channels in Placentas from Normal and Pathological Pregnancies: Targeting of the K<Subscript>ir</Subscript> 2.1 Channel to Lipid Rafts

Potassium channels play important physiological roles in human syncytiotrophoblasts (hSTBs) from placenta, an epithelium responsible for maternal–fetal exchange. Basal and apical plasma membranes differ in their lipid and protein composition, and the latter contains cholesterol-enriched microdomains. In placental tissue, the specific localization of potassium channels is unknown. Previously, we described two isolated subdomains from the apical membrane (MVM and LMVM) and their respective microdomains (lipid rafts). Here, we report on the distribution of K_ir2.1, K_v2.1, TASK-1, and TREK-1 in hSTB membranes and the lipid rafts that segregate them. Immunoblotting experiments showed that these channels are present mainly in the apical membrane from healthy hSTBs. Apical expression versus basal membrane was 84 and 16% for K_ir2.1 and K_v2.1, 60 and 30% for TREK-1, and 74 and 26% for TASK-1. Interestingly, K_v2.1 showed differences between apical membrane subdomains: 26 ± 8% was located in the LMVM and 59 ± 9% in MVM. In pathological placentas, the expression distribution changed in the basal membrane: preeclampsia shifted to 50% and intrauterine growth restriction to 42% for TASK-1 and both pathologies increased to 25% for K_ir2.1 and K_v2.1, K_ir2.1 appeared to be associated with rafts that were sensitive to cholesterol depletion in healthy, but not in pathological, placentas. K_v2.1 and TREK-1 emerged in the nonraft fractions. The precise membrane localization of ion channels in hSTB membranes is necessary to understand the physiological events.     

β-Adrenergic Modulation of Glial Inwardly Rectifying Potassium Channels

Abstract: Cultured spinal cord astrocytes (2–13 days in vitro) express several different potassium current types, including delayed rectifier, transient A-type, and inward rectifier (K_ir) K⁺ currents. Of these, K_ir is believed to be of critical importance in the modulation of extracellular [K⁺] in the CNS. Using the whole-cell patch-clamp technique, we analyzed modulation of K_ir currents by β-adrenergic receptor activation. The selective β-adrenergic agonist isoproterenol (1–100 µM) and epinephrine (1–100 µM) each reduced peak K_ir current amplitudes to 52.7 ± 12.5 and 63.6 ± 7.0%, respectively, at 100 µM. Forskolin (K_D of ～25 µM), an activator of adenylate cyclase (AC), and dibutyryl-cyclic AMP (1 mM), a membrane-permeable analogue of cyclic AMP (cAMP), were each used to increase [cAMP]_i, the product of AC, and resulted in similar reductions of K_ir currents. By contrast, 1,9-dideoxyforskolin (1–50 µM), a forskolin analogue that does not activate AC, did not affect K_ir currents, indicating that AC activity is a required element for K_ir modulation. Three inhibitors of PKA—Rp-adenosine 3′,5′-cyclic monophosphothioate, H-7, and adenosine 3′,5′-cyclic monophosphate-dependent protein kinase inhibitor—failed to inhibit K_ir current reduction by β-adrenergic agonists. These results indicate that β-adrenergic receptor ligands can modulate K_ir currents and suggest that this modulation involves activation of AC but not protein kinase A. Such modulation may provide a mechanism by which neurons can modulate glial K_ir currents and thereby may affect glial K⁺“spatial buffering” in the CNS.     

Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K⁺ channels (Kir) control extracellular [K⁺]_o homeostasis by uptake of K⁺ from the extracellular space and release of K⁺ into the microvasculature. Kir channels were also recently implicated in K⁺-associated water influx and cell swelling. We studied the time-dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co-localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time-lapse-two-photon laser-scanning microscopy in situ . Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co-application of hypotonic solution and Ba²⁺, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co-application with Ba²⁺. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.     

Bistability dynamics in simulations of neural activity in high-extracellular-potassium conditions

Modulation of extracellular potassium concentration ([K]_o) has a profound impact on the excitability of neurons and neuronal networks. In the CA3 region of the rat hippocampus synchronized epileptiform bursts occur in conditions of increased [K]_o. The dynamic nature of spontaneous neuronal firing in high [K]_o is therefore of interest. One particular interest is the potential presence of bistable behaviors such as the coexistence of stable repetitive firing and fixed rest potential states generated in individual cells by the elevation of [K]_o. The dynamics of repetitive activity generated by increased [K]_o is investigated in a 19-compartment hippocampal pyramidal cell (HPC) model and a related two-compartment reduced HPC model. Results are compared with those for the Hodgkin-Huxley equations in similar conditions. For neural models, [K]_o changes are simulated as a shift in the potassium reversal potential (E _K ). Using phase resetting and bifurcation analysis techniques, all three models are shown to have specific regions of E _K that result in bistability. For activity in bistable parameter regions, stimulus parameters are identified that switch high-potassium model behavior from repetitive firing to a quiescent state. Bistability in the HPC models is limited to a very small parameter region. Consequently, our results suggest that it is likely some HPCs in networks exposed to high [K]_o continue to burst such that a stable, quiescent network state does not exist. In [K]_o ranges where HPCs are not bistable, the population may still exhibit bistable behaviors where synchronous population events are reversibly annihilated by phase resetting pulses, suggesting the existence of a nonsynchronous network attractor.     

Modeling the effects of extracellular potassium on bursting properties in pre-Bötzinger complex neurons

There are many types of neurons that intrinsically generate rhythmic bursting activity, even when isolated, and these neurons underlie several specific motor behaviors. Rhythmic neurons that drive the inspiratory phase of respiration are located in the medullary pre-Bötzinger Complex (pre-BötC). However, it is not known if their rhythmic bursting is the result of intrinsic mechanisms or synaptic interactions. In many cases, for bursting to occur, the excitability of these neurons needs to be elevated. This excitation is provided in vitro (e.g. in slices), by increasing extracellular potassium concentration (K _out ) well beyond physiologic levels. Elevated K _out shifts the reversal potentials for all potassium currents including the potassium component of leakage to higher values. However, how an increase in K _out , and the resultant changes in potassium currents, induce bursting activity, have yet to be established. Moreover, it is not known if the endogenous bursting induced in vitro is representative of neural behavior in vivo. Our modeling study examines the interplay between K _out , excitability, and selected currents, as they relate to endogenous rhythmic bursting. Starting with a Hodgkin-Huxley formalization of a pre-BötC neuron, a potassium ion component was incorporated into the leakage current, and model behaviors were investigated at varying concentrations of K _out . Our simulations show that endogenous bursting activity, evoked in vitro by elevation of K _out , is the result of a specific relationship between the leakage and voltage-dependent, delayed rectifier potassium currents, which may not be observed at physiological levels of extracellular potassium.     

Regulation of voltage-gated potassium channels by PI(4,5)P2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) regulates activities of numerous ion channels including inwardly rectifying potassium (K_ir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K_V) channels might be regulated by PI(4,5)P₂. Wide expression of K_V channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K_V channels by PI(4,5)P₂, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M₁R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P₂ with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P₂ at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K_V7.1, K_V7.2/7.3, and K_ir2.1 channel current by 90–95%. Activation of M₁R inhibited K_V7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P₂ regulation of activity of K_V1.1/K_Vβ1.1, K_V1.3, K_V1.4, and K_V1.5/K_Vβ1.3, K_V2.1, K_V3.4, K_V4.2, K_V4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K_V1.1/K_Vβ1.1 and K_V3.4, resulting in up-regulation of current density upon activation of M₁R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P₂ at the plasma membrane by enzymes does not seem to influence activity of most tested K_V channels, whereas it does strongly inhibit members of the K_V7 and K_ir families.     

Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.     

The Nucleotide-Binding Sites of SUR1: A Mechanistic Model

ATP-sensitive potassium (K_ATP) channels comprise four pore-forming Kir6.2 subunits and four modulatory sulfonylurea receptor (SUR) subunits. The latter belong to the ATP-binding cassette family of transporters. K_ATP channels are inhibited by ATP (or ADP) binding to Kir6.2 and activated by Mg-nucleotide interactions with SUR. This dual regulation enables the K_ATP channel to couple the metabolic state of a cell to its electrical excitability and is crucial for the K_ATP channel’s role in regulating insulin secretion, cardiac and neuronal excitability, and vascular tone. Here, we review the regulation of the K_ATP channel by adenine nucleotides and present an equilibrium allosteric model for nucleotide activation and inhibition. The model can account for many experimental observations in the literature and provides testable predictions for future experiments.     

Inwardly rectifying potassium channels (Kir) in central nervous system glia: a special role for Kir4.1 in glial functions

Glia in the central nervous system (CNS) express diverse inward rectifying potassium channels (Kir). The major function of Kir is in establishing the high potassium (K+) selectivity of the glial cell membrane and strongly negative resting membrane potential (RMP), which are characteristic physiological properties of glia. The classical property of Kir is that K+ flows inwards when the RMP is negative to the equilibrium potential for K+ (E(K)), but at more positive potentials outward currents are inhibited. This provides the driving force for glial uptake of K+ released during neuronal activity, by the processes of "K+ spatial buffering" and "K+ siphoning", considered a key function of astrocytes, the main glial cell type in the CNS. Glia express multiple Kir channel subtypes, which are likely to have distinct functional roles related to their differences in conductance, and sensitivity to intracellular and extracellular factors, including pH, ATP, G-proteins, neurotransmitters and hormones. A feature of CNS glia is their specific expression of the Kir4.1 subtype, which is a major K+ conductance in glial cell membranes and has a key role in setting the glial RMP. It is proposed that Kir4.1 have a primary function in K+ regulation, both as homomeric channels and as heteromeric channels by co-assembly with Kir5.1 and probably Kir2.0 subtypes. Significantly, Kir4.1 are also expressed by oligodendrocytes, the myelin-forming cells of the CNS, and the genetic ablation of Kir4.1 results in severe hypomyelination. Hence, Kir, and in particular Kir4.1, are key regulators of glial functions, which in turn determine neuronal excitability and axonal conduction.