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During neuronal activity in the mammalian brain, the K+ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na+ /K+ /2Cl- cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity-dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion-sensitive microelectrodes to measure changes in ECS, [K+ ]o and [H+ ]o /pHo during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K+ and glutamate released into the extracellular space (the K+ /Cl- cotransporter, KCC, glutamate transporters and G protein-coupled receptors) did not modulate the extracellular space dynamics. The HCO3--transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na+ / HCO3- cotransporter 1 (NBCe1), is activated by the K+ -mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by ∼25% without affecting the K+ transients, pointing to NBCe1 as a key contributor to the stimulus-induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like-wise, reduced the ECS shrinkage by ∼25% without compromising the K+ transients. Isosmotic reduction of extracellular Cl- revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus-evoked astrocytic cell swelling does not appear to occur as a direct effect of the K+ clearance, as earlier proposed, but partly via the pH-regulating transport mechanisms activated by the K+ -induced astrocytic depolarization and the activity-dependent metabolism.

Key points Mammalian cells are frequently exposed to stressors causing volume changes. The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux. The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive. TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins. TRPV4 functions as a volume-sensing ion channel irrespective of the origin of the cell swelling. Abstract Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non-selective cation channel permeable to Ca2+, it transduces physical stress in the form of osmotic cell swelling into intracellular Ca2+-dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4–AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume-sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co-expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4-mediated transmembrane currents. Similar TRPV4 activation was induced by co-expression of a cognate channel, AQP1. The level of osmotically-induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co-expressed water-translocating Na+/K+/2Cl− cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume-dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume-sensor, rather than as an osmo-sensor.

Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cAMP dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling in AQP2 membrane targeting in Madin-Darby canine kidney and mouse cortical collecting duct (mpkCCD14) cells was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost), and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP2 membrane targeting, but did not significantly increase cAMP levels. After washout of the EP2 agonist or dDAVP, AQP2 membrane abundance remained elevated for at least 80 min, whereas cAMP levels rapidly decreased. Similar effects of the EP2 agonist were also observed for AQP2 constitutively nonphosphorylated at ser-269. The adenylyl cyclase inhibitor SQ22536 did not prevent AQP2 targeting during stimulation of each receptor, nor after dDAVP washout. In conclusion, this study demonstrates that although direct stimulation with cAMP causes AQP2 membrane targeting, cAMP is not necessary for receptor-mediated AQP2 membrane targeting and Gs-coupled receptors can also signal through an alternative pathway that increases AQP2 membrane targeting.

KEY POINTS The large-pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C-terminal cleavage led to a Panx1 open-state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1-mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. ABSTRACT Pannexin 1 (Panx1) is a large-pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant-selectivity of Panx1 activation and inhibition, we employed Panx1-expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C-terminal truncation or cell shrinkage elevated Panx1-mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K+ ]o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl- , lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1-mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant-selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.

Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.

Correction Volume: 143 Pages: 349-350 DOI: 10.1016/j.neuropharm.2018.10.012 Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.

Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient

KEY POINTS Management of glutamate and K+ in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na+ /K+ -ATPase isoform combination is activated by the K+ transients occurring during neuronal activity. In the present study, we report that glutamate transporter-mediated astrocytic Na+ transients stimulate the Na+ /K+ -ATPase and thus the clearance of extracellular K+ . Specifically, the astrocytic α2β1 Na+ /K+ -ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+ . Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+ /K+ -ATPase β-subunits. ABSTRACT Neuronal activity is associated with transient [K+ ]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+ /K+ -ATPase of which several subunit isoform combinations exist. The astrocytic Na+ /K+ -ATPase α2β2 isoform constellation responds directly to increased [K+ ]o but, in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+ /K+ -ATPase was not fully saturated at basal astrocytic [Na+ ]i , irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i transients associated with activity-induced glutamate transporter activity.

A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K+, Cl-, and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl- channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl- currents and AVD, but it remains unclear whether these anoctamins operate as Cl- channels or as regulators of other apoptotic Cl- channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling.

The Na(+)-K(+)-Cl(-) cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl(-) concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure-function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC50s) of bumetanide were 0.68 (hNKCC1A) and 4.0μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC50s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1.

Background: Cerebral edema can cause life-threatening increase in intracranial pressure. Besides surgical craniectomy performed in severe cases, osmotherapy may be employed to lower the intracranial pressure by osmotic extraction of cerebral fluid upon intravenous infusion of mannitol or NaCl. A so-called rebound effect can, however, hinder continuous reduction in cerebral fluid by yet unresolved mechanisms. Methods: We determined the brain water and electrolyte content in healthy rats treated with osmotherapy. Osmotherapy (elevated plasma osmolarity) was mediated by intraperitoneal injection of NaCl or mannitol with inclusion of pharmacological inhibitors of selected ion-transporters present at the capillary lumen or choroidal membranes. Brain barrier integrity was determined by fluorescence detection following intravenous delivery of Na+-fluorescein. Results: NaCl was slightly more efficient than mannitol as an osmotic agent. The brain water loss was only ∼ 60% of that predicted from ideal osmotic behavior, which could be accounted for by cerebral Na+ and Cl- accumulation. This electrolyte accumulation represented the majority of the rebound response, which was unaffected by the employed pharmacological agents. The brain barriers remained intact during the elevated plasma osmolarity. Conclusions: A brain volume regulatory response occurs during osmotherapy, leading to the rebound response. This response involves brain accumulation of Na+ and Cl- and takes place by unresolved molecular mechanisms that do not include the common ion-transporting mechanisms located in the capillary endothelium at the blood-brain barrier and in the choroid plexus epithelium at the blood-CSF barrier. Future identification of these ion-transporting routes could provide a pharmacological target to prevent the rebound effect associated with the widely used osmotherapy.

In the original version of this Article, the affiliation for Lorena Rojas-Vega and Gerardo Gamba was incomplete. The correct affiliation for these authors appears below. Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autonoma de México and Instituto Nacional de Ciencias Médicas y Nutricion Salvador Zubiran, Mexico City, Mexico. This error has now been corrected in the PDF and HTML versions of the Article.

More than 90% of the cases of Parkinson’s disease have unknown etiology. Gradual loss of dopaminergic neurons of substantia nigra is the main cause of morbidity in this disease. External factors such as environmental toxins are believed to play a role in the cell loss, although the cause of the selective vulnerability of dopaminergic neurons remains unknown. We have previously shown that aquaglyceroporin AQP9 is expressed in dopaminergic neurons and astrocytes of rodent brain. AQP9 is permeable to a broad spectrum of substrates including purines, pyrimidines, and lactate, in addition to water and glycerol. Here we test our hypothesis that AQP9 serves as an influx route for exogenous toxins and, hence, may contribute to the selective vulnerability of nigral dopaminergic (tyrosine hydroxylase-positive) neurons. Using Xenopus oocytes injected with Aqp9 cRNA, we show that AQP9 is permeable to the parkinsonogenic toxin 1-methyl-4-phenylpyridinium (MPP+). Stable expression of AQP9 in HEK cells increases their vulnerability to MPP+ and to arsenite—another parkinsonogenic toxin. Conversely, targeted deletion of Aqp9 in mice protects nigral dopaminergic neurons against MPP+ toxicity. A protective effect of Aqp9 deletion was demonstrated in organotypic slice cultures of mouse midbrain exposed to MPP+ in vitro and in mice subjected to intrastriatal injections of MPP+ in vivo. Seven days after intrastriatal MPP+ injections, the population of tyrosine hydroxylase-positive cells in substantia nigra is reduced by 48% in Aqp9 knockout mice compared with 67% in WT littermates. Our results show that AQP9 –selectively expressed in catecholaminergic neurons—is permeable to MPP+ and suggest that this aquaglyceroporin contributes to the selective vulnerability of nigral dopaminergic neurons by providing an entry route for parkinsonogenic toxins. To our knowledge this is the first evidence implicating a toxin permeable membrane channel in the pathophysi-ology of Parkinson’s disease.

Aquaporin-4, encoded by AQP4, is the major water channel in the central nervous system and plays an important role in the brain's water balance, including edema formation and clearance. Using genomic copy-number analysis and trio-exome sequencing, we investigated a male patient with intellectual disability, hearing loss, and progressive gait dysfunction and found a de novo missense change Ser111Thr in AQP4 as the only suspicious finding. Perinatally, signs of brain ischemia were detected in relation to acute collapse 2 h after birth that resolved a few days later. At the age of 3 mo, cardiac hypertrophy was detected that persisted through childhood but was completely resolved by age 16. In theory, this neurodevelopmental disorder with transient cardiomyopathy could be caused by a disturbance of cellular water balance. Ser111 is an extremely conserved residue in the short cytoplasmic loop between AQP4 transmembrane helix 2 and 3, present across all AQP isoforms from plants to mammals, and it does not appear to be a phosphorylation site. We found that the Ser111Thr change does not affect water permeability or protein stability, suggesting another and possibly regulatory role. Although causality remains unproven, this case study draws attention to AQP4 as a candidate gene for a unique developmental disorder and to a specific serine as a residue of possibly great functional importance in many AQPs.

Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas fromTrpv4−/−andAqp4−/−mice exhibited suppressed transcription of genes encodingTrpv4,Aqp4, and theKir4.1subunit of inwardly rectifying potassium channels. Swelling and [Ca2+]ielevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) orTrpv4ablation. Elimination ofAqp4suppressed swelling-induced [Ca2+]ielevations but only modestly attenuated the amplitude of Ca2+signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressingXenopusoocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca2+entry through TRPV4 channels reciprocally modulates volume regulation, swelling, andAqp4gene expression. Therefore, TRPV4–AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling.SIGNIFICANCE STATEMENTWe characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression ofAqp4andKir4.1genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation.

Spreading depolarizations (SDs) are waves of sustained neuronal and glial depolarization that propagate massive disruptions of ion gradients through the brain. SD is associated with migraine aura and recently recognized as a novel mechanism of injury in stroke and brain trauma patients. SD leads to neuronal swelling as assessed in real time with two-photon laser scanning microscopy (2PLSM). Pyramidal neurons do not express aquaporins and thus display low inherent water permeability, yet SD rapidly induces focal swelling (beading) along the dendritic shaft by unidentified molecular mechanisms. To address this issue, we induced SD in murine hippocampal slices by focal KCl microinjection and visualized the ensuing beading of dendrites expressing EGFP by 2PLSM. We confirmed that dendritic beading failed to arise during large (100 mOsm) hyposmotic challenges, underscoring that neuronal swelling does not occur as a simple osmotic event. SD-induced dendritic beading was not prevented by pharmacological interference with the cytoskeleton, supporting the notion that dendritic beading may result entirely from excessive water influx. Dendritic beading was strictly dependent on the presence of Cl−, and, accordingly, combined blockade of Cl−-coupled transporters led to a significant reduction in dendritic beading without interfering with SD. Furthermore, ourin vivodata showed a strong inhibition of dendritic beading during pharmacological blockage of these cotransporters. We propose that SD-induced dendritic beading takes place as a consequence of the altered driving forces and thus activity for these cotransporters, which by transport of water during their translocation mechanism may generate dendritic beading independently of osmotic forces.SIGNIFICANCE STATEMENTSpreading depolarization occurs during pathological conditions such as stroke, brain injury, and migraine and is characterized as a wave of massive ion translocation between intracellular and extracellular space in association with recurrent transient focal swelling (beading) of dendrites. Numerous ion channels have been demonstrated to be involved in generation and propagation of spreading depolarization, but the molecular machinery responsible for the dendritic beading has remained elusive. Using real-timein vitroandin vivotwo-photon laser scanning microscopy, we have identified the transport mechanisms involved in the detrimental focal swelling of dendrites. These findings have clear clinical significance because they may point to a new class of pharmacological targets for prevention of neuronal swelling that consequently will serve as neuroprotective agents.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. Based on studies on AQP4(-/-) mice, AQP4 has been assigned physiological roles in stimulus-induced K(+) clearance, paravascular fluid flow, and brain edema formation. Conflicting data have been presented on the role of AQP4 in K(+) clearance and associated extracellular space shrinkage and on the stroke-induced alterations of AQP4 expression levels during edema formation, raising questions about the functional importance of AQP4 in these (patho)physiological aspects. Phosphorylation-dependent gating of AQP4 has been proposed as a regulatory mechanism for AQP4-mediated osmotic water transport. This paradigm was, however, recently challenged by experimental evidence and molecular dynamics simulations. Regulatory patterns and physiological roles for AQP4 thus remain to be fully explored.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser276, Ser285, Ser315, Ser316, Ser321, and Ser322. To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel.

The blood–brain barrier (BBB) is involved in brain water and salt homeostasis. Blood osmolarity increases during dehydration and water is osmotically extracted from the brain. The loss of water is less than expected from pure osmotic forces, due to brain electrolyte accumulation. Although the underlying molecular mechanisms are unresolved, the current model suggests the luminally expressed Na(+)-K(+)-2Cl(−) co-transporter 1 (NKCC1) as a key component, while the role of the Na(+)/K(+)-ATPase remains uninvestigated. To test the involvement of these proteins in brain electrolyte flux under mimicked dehydration, we employed a tight in vitro co-culture BBB model with primary cultures of brain endothelial cells and astrocytes. The Na(+)/K(+)-ATPase and the NKCC1 were both functionally dominant in the abluminal membrane. Exposure of the in vitro BBB model to conditions mimicking systemic dehydration, i.e. hyperosmotic conditions, vasopressin, or increased [K(+)](o) illustrated that NKCC1 activity was unaffected by exposure to vasopressin and to hyperosmotic conditions. Hyperosmotic conditions and increased K(+) concentrations enhanced the Na(+)/K(+)-ATPase activity, here determined to consist of the α1β1 and α1β3 isozymes. Abluminally expressed endothelial Na(+)/K(+)-ATPase, and not NKCC1, may therefore counteract osmotic brain water loss during systemic dehydration by promoting brain Na(+) accumulation.

The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na+ and K+ homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimulation. Thiazide-sensitive sodium-22 uptakes were elevated in the mutants and internalization from the plasma membrane was significantly less than WT-NCC. K909R had increased half-life, whereas chloroquine or MG132 treatment indicated that K706 and K909 play roles in lysosomal and proteasomal NCC degradation, respectively. In conclusion, site-specific ubiquitylation of NCC plays alternative roles for NCC function.

Synaptic activity results in transient elevations in extracellular K

KEY POINTS: Mammalian cells are frequently exposed to stressors causing volume changes. The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux. The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive. TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins. TRPV4 functions as a volume‐sensing ion channel irrespective of the origin of the cell swelling. ABSTRACT: Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non‐selective cation channel permeable to Ca(2+), it transduces physical stress in the form of osmotic cell swelling into intracellular Ca(2+)‐dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4–AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume‐sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co‐expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4‐mediated transmembrane currents. Similar TRPV4 activation was induced by co‐expression of a cognate channel, AQP1. The level of osmotically‐induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co‐expressed water‐translocating Na(+)/K(+)/2Cl(−) cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume‐dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume‐sensor, rather than as an osmo‐sensor.

Neuronal activity results in release of K(+) into the extracellular space of the central nervous system. If the excess K(+) is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K(+) from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K(+) has been promoted as a major contributor to K(+) removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K(+) management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K(+) during but not after neuronal activity.

Network activity in the brain is associated with a transient increase in extracellular K(+) concentration. The excess K(+) is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na(+)/K(+)/2Cl(-) cotransporter 1 (NKCC1), and/or Na(+)/K(+)-ATPase activity. Their individual contribution to [K(+)]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na(+)/K(+)-ATPase and to resolve their involvement in clearance of extracellular K(+) transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K(+)]o increases above basal levels. Increased [K(+)]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K(+) clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K(+) removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K(+)]o increase. In contrast, inhibition of the different isoforms of Na(+)/K(+)-ATPase reduced post-stimulus clearance of K(+) transients. The astrocyte-characteristic α2β2 subunit composition of Na(+)/K(+)-ATPase, when expressed in Xenopus oocytes, displayed a K(+) affinity and voltage-sensitivity that would render this subunit composition specifically geared for controlling [K(+)]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na(+)/K(+)-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K(+) recovery in native hippocampal tissue while Kir4.1 and Na(+)/K(+)-ATPase serve temporally distinct roles.

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a 'docked' counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.

Key points • We test a novel model of osmosis in aquaporins. • A solute molecule present at the pore mouth can be reflected or permeate the pore; we propose that only reflected molecules induce osmotic water transport, while permeating molecules give rise to no water transport. • We tested a range of channel geometries using aquaporins AQP1 and AQP9 and mutants thereof; the aquaporins were expressed in Xenopus oocytes. Osmotic gradients were generated by solutes of molecular weights in the range 45–595 Daltons. The reflected fraction of a given solute was estimated optically and compared to the permeability obtained from uptakes of radio-labelled solutes. • In accordance with our model there was a linear relationship between solute permeability and reflection coefficient: solutes with high permeability had low reflection coefficients and vice versa. • We found no evidence for significant coupling between water and solute fluxes inside the pore. Abstract We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, PS, is proportional to 1 –σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mm of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short-term uptake of radio-labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward-facing end of the pore.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser(111) to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser(111) and of phosphorylation-dependent gating of AQP4.

During neuronal activity in the brain, extracellular K(+) rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K(+) is the Na(+)/K(+)-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na(+)/K(+)-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K(+) from neurons, whereas the neurons themselves become the primary K(+) absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na(+)/K(+)-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K(+) from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na(+)/K(+)-ATPase isoform combinations in K(+) management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na(+)/K(+)-ATPase in the regulation of extracellular K(+) in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of σ, all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3β and -ζ, whereas levels of 14-3-3η and -θ were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2/14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3ζ and -θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3θ and -ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation.

A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K

Tubulointerstitial nephritis is a common cause of kidney failure and may have diverse etiologies. This form of nephritis is sometimes associated with autoimmune disease, but the role of autoimmunemechanisms in disease development is not well understood. Here, we present the cases of three patients with autoimmune polyendocrine syndrome type 1 who developed tubulointerstitial nephritis and ESRD in association with autoantibodies against kidney collecting duct cells. One of the patients developed autoantibodies targeting the collecting duct-specificwater channel aquaporin 2, whereas autoantibodies of the two other patients reacted against the HOXB7 or NFAT5 transcription factors, which regulate the aquaporin 2 promoter. Our findings suggest that tubulointerstitial nephritis developed in these patients as a result of an autoimmune insult on the kidney collecting duct cells.

The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antibodies targeting pS124-NCC demonstrated a band of 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with kidney tubule segment-specific markers localized pS124-NCC to all distal convoluted tubule cells. Double immunogold electron microscopy demonstrated that pS124-NCC co-localized with total NCC in the apical plasma membrane of distal convoluted tubule cells and intracellular vesicles. Acute treatment of Munich-Wistar rats or vasopressin-deficient Brattleboro rats with the vasopressin type 2 receptor-specific agonist dDAVP significantly increased pS124-NCC abundance, with no changes in total NCC plasma membrane abundance. pS124-NCC levels also increased in abundance in rats after stimulation of the renin-angiotensin-aldosterone system by dietary low sodium intake. In contrast to other NCC phosphorylation sites, the STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and mTOR/FRAP) showed weak but significant phosphorylation activity at S124. In oocytes, (36)Cl uptake studies combined with biochemical analysis showed decreased activity of plasma membrane-associated NCC when replacing S124 with alanine (A) or aspartic acid (D). In novel tetracycline-inducible MDCKII-NCC cell lines, S124A and S124D mutants were able to traffic to the plasma membrane similarly to wildtype NCC.

Water transport across cell membranes is central to most physiological functions. About 200 L of water move across epithelial cells each day in humans in order to maintain whole-body homeostasis; water transport in and out of organs such as the brain and eye are of major clinical importance. It is well established that the water transport is driven by ion transport, but how? Osmosis is not always the answer: water can be transported against considerable osmotic gradients, apparently without any external osmotic or hydrostatic driving forces. It is generally accepted that cotransporters of the symport type play a key role for the coupling between ion and water fluxes. Models of coupling are either molecular or based on unstirred layer effects, and can be distinguished by their response time: for molecular models, water transport follows changes of substrate transport instantaneously; in unstirred layer models there is a delay while the osmolarity changes in the solutions surrounding the cotransport protein. For cotransporters expressed heterologously in Xenopus oocytes, influx of water can be detected about 1 second after initiation of cotransport of ions and other substrates. This is 20 times faster than expected (and observed) for unstirred layer effects. Water transport in cotransporters is best explained by a molecular model in which ion and water fluxes are coupled by a mechanism within the protein. This would also clarify how cotransporters exploit the free energy in the ion fluxes for the uphill transport of water. WIREs Membr Transp Signal 2012, 1:373–381. doi: 10.1002/wmts.54 For further resources related to this article, please visit the WIREs website.

Increased ammonium (NH4+/NH3) in the brain is a significant factor in the pathophysiology of hepatic encephalopathy, which involves altered glutamatergic neurotransmission. In glial cell cultures and brain slices, glutamate uptake either decreases or increases following acute ammonium exposure but the factors responsible for the opposing effects are unknown. Excitatory amino acid transporter isoforms EAAT1, EAAT2, and EAAT3 were expressed in Xenopus oocytes to study effects of ammonium exposure on their individual function. Ammonium increased EAAT1- and EAAT3-mediated [3H]glutamate uptake and glutamate transport currents but had no effect on EAAT2. The maximal EAAT3-mediated glutamate transport current was increased but the apparent affinities for glutamate and Na+ were unaltered. Ammonium did not affect EAAT3-mediated transient currents, indicating that EAAT3 surface expression was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH3 mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte cytoplasm, 2) intracellular pH buffering with MOPS did not reduce the stimulation, and 3) ammonium enhanced pH-independent cysteine transport. Our data suggest that the ammonium-elicited uptake stimulation is not caused by intracellular alkalinization or changes in the concentrations of cotransported ions but may be due to a direct effect on EAAT1/EAAT3. We predict that EAAT isoform-specific effects of ammonium combined with cell-specific differences in EAAT isoform expression may explain the conflicting reports on ammonium-induced changes in glial glutamate uptake.

The NKCC1 and NKCC2 isoforms of the mammalian Na+–K+–2Cl− cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined. Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic increases in external K+ concentration elicited abrupt inward water fluxes in NKCC1; the K+ dependence obeyed one-site kinetics with a K0.5 of 7.5 mm. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l−1. A comparison between ion and water fluxes indicates that 460 water molecules are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes. Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the notion of interaction between ions and water in NKCC1 and allows for an estimate of around 600 water molecules transported per turnover of the protein. Osmotic gradients did not induce water transport in NKCC2. We conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfils its role in the kidney of transporting ions but not water. The different behaviour of NKCC1 and NKCC2 is discussed on the basis of recent molecular models based on studies of structural and molecular dynamics.

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K(+) concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.

The GABA transporter-1 (GAT-1) has three current-generating modes: GABA-coupled current, Li+-induced leak current, and Na+-dependent transient currents. We earlier hypothesized that Li+ is able to substitute for the first Na+ in the transport cycle and thereby induce a distinct conformation in GAT-1 and that the onset of the Li+-induced leak current at membrane potentials more negative than −50 mV was due to a voltage-dependent conformational change of the Li+-bound transporter. In this study, we set out to verify this hypothesis and seek insight into the structural dynamics underlying the leak current, as well as the sodium-dependent transient currents, by applying voltage clamp fluorometry to tetramethylrhodamine 6-maleimide-labeled GAT-1 expressed in Xenopus laevis oocytes. MTSET accessibility studies demonstrated the presence of two distinct conformations of GAT-1 in the presence of Na+ or Li+. The voltage-dependent fluorescence intensity changes obtained in Li+ buffer correlated with the Li+-induced leak currents, i.e. both were highly voltage-dependent and only present at hyperpolarized potentials (

It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH 4 + or methylammonium ions (MeA+). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH 4 + or MeA+ transport. This was confirmed by the close correlation between the uptake of radio-labeled MeA+ and MeA+-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH 4 + and MeA+. We suggest a model where NH 4 + is transported as NH3 and H+ via separate pathways but the latter two recombine before leaving the protein.

Udgivelsesdato: 2009-Mar Arginine vasopressin (AVP)-regulated phosphorylation of the water channel aquaporin-2 (AQP2) at serine 256 (S256) is essential for its accumulation in the apical plasma membrane of collecting duct principal cells. In this study, we examined the role of additional AVP-regulated phosphorylation sites in the COOH-terminal tail of AQP2 on protein function. When expressed in Xenopus laevis oocytes, prevention of AQP2 phosphorylation at S256A (S256A-AQP2) reduced osmotic water permeability threefold compared with wild-type (WT) AQP2-injected oocytes. In contrast, prevention of AQP2 single phosphorylation at S261 (S261A), S264 (S264A), and S269 (S269A), or all three sites in combination had no significant effect on water permeability. Similarly, oocytes expressing S264D-AQP2 and S269D-AQP2, mimicking AQP2 phosphorylated at these residues, had similar water permeabilities to WT-AQP2-expressing oocytes. The use of high-resolution confocal laser-scanning microscopy, as well as biochemical analysis demonstrated that all AQP2 mutants, with the exception of S256A-AQP2, had equal abundance in the oocyte plasma membrane. Correlation of osmotic water permeability relative to plasma membrane abundance demonstrated that lack of phosphorylation at S256, S261, S264, or S269 had no effect on AQP2 unit water transport. Similarly, no effect on AQP2 unit water transport was observed for the 264D and 269D forms, indicating that phosphorylation of the COOH-terminal tail of AQP2 is not involved in gating of the channel. The use of phosphospecific antibodies demonstrated that AQP2 S256 phosphorylation is not dependent on any of the other phosphorylation sites, whereas S264 and S269 phosphorylation depend on prior phosphorylation of S256. In contrast, AQP2 S261 phosphorylation is independent of the phosphorylation status of S256.

The glucose transporter GLUT2 has been shown to also transport water. In the present paper we investigated the relation between sugar and water transport in human GLUT2 expressed in Xenopus oocytes. Sugar transport was determined from uptakes of non-metabolizable glucose analogues, primarily 3-O-methyl-d-glucopyranoside; key experimental results were confirmed using d(+)-glucose. Water transport was derived from changes in oocyte volume monitored at a high resolution (20 pl, 1 s). Expression of GLUT2 induced a sugar permeability, PS, of about 5 × 10−6 cm s−1 and a passive water permeability, Lp, of 5.5 × 10−5 cm s−1. Accordingly, the passive water permeability of a GLUT2 protein is about 10 times higher than its sugar permeability. Both permeabilities were abolished by phloretin. Isosmotic addition of sugar to the bathing solution (replacing mannitol) induced two parallel components of water influx in GLUT2, one by osmosis and one by cotransport. The osmotic driving force arose from sugar accumulation at the intracellular side of the membrane and was given by an intracellular diffusion coefficient for sugar of 10−6 cm2 s−1, one-fifth of the free solution value. The diffusion coefficient was determined in oocytes coexpressing GLUT2 and the water channel AQP1 where water transport was predominantly osmotic. By the cotransport mechanism about 35 water molecules were transported for each sugar molecule by a mechanism within the GLUT2. These water molecules could be transported uphill, against an osmotic gradient, energized by the flux of sugar. This capacity for cotransport is 10 times smaller than that of the Na+-coupled glucose transporters (SGLT1). The physiological role of GLUT2 for intestinal transport under conditions of high luminal sugar concentrations is discussed.

Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC.

The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K(+) current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4-2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems.

Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results.

Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca2+]ielevations that were inhibited by TRPV4 antagonists and absent inTRPV4−/−Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca2+waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca2+signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function.

It isgenerally accepted that cotransporters transport water in addition to their normal substrates, although the pre- cise mechanism is debated; both active and passive modes of transport have been suggested. The magnitude of the waterflux mediated by cotransporters may well be significant: both the number of cotransporters per cell and the unit water permeabil- ity are high. For example, the Na-glutamate cotransporter (EAAT1) has a unit water permeability one tenth of that of aquaporin (AQP) 1. Cotransporters are widely distributed in the brain and participate in several vital functions: inorganic ions are transported by K-Cl and Na-K-Cl cotransporters, neurotransmitters are reabsorbed from the synaptic cleft by Na-dependent cotransporters located on glial cells and neu- rons, and metabolites such as lactate are removed from the extracellular space by means of H-lactate cotransporters. We have previously determined water transport capacities for these cotransporters in model systems (Xenopus oocytes, cell cul- tures, and in vitro preparations), and will discuss their role in water homeostasis of the astroglial cell under both normo- and pathophysiologal situations. Astroglia is a polarized cell with EAAT localized at the end facing the neuropil while the end abutting the circulation is rich in AQP4. The water transport properties of EAAT suggest a new model for volume homeosta- sis of the extracellular space during neural activity. © 2004 This review is focused on cotransporters that are ex- pressed in the brain and have been found to transport water: the KCC (K-Cl), the NKCC (Na-K-Cl), the MCT (H-lactate), the GAT (Na-GABA), and the EAAT (Na-glutamate),forrecentreviewssee(ZeuthenandMac- Aulay, 2002a,b). We will review the localization and phys- iological function of these cotransporters and estimate their contribution to the membrane water permeability con- sidering their high level of expression and relatively high unit water permeability. Specifically, we will discuss the role of the unilateral distribution of cotransporters toward the neuropil aspects of the astroglial cell. The transport properties of this part of the membrane have a central role for the function of the synapse, since they control the dimensions and composition of the extracellular space. The water transport properties of the astroglial cell mem- brane facing the neuropil are determined by cotransport- ers, in particular EAAT and NKCC, since no other water

Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X7 yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca(2+) and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X7, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.

Two high affinity Zn2+ binding sites were engineered in the otherwise Zn2+-insensitive rat γ-aminobutyric acid (GABA) transporter-1 (rGAT-1) based on structural information derived from Zn2+ binding sites engineered previously in the homologous dopamine transporter. Introduction of a histidine (T349H) at the extracellular end of transmembrane segment (TM) 7 together with a histidine (E370H) or a cysteine (Q374C) at the extracellular end of TM 8 resulted in potent inhibition of [3H]GABA uptake by Zn2+ (IC50 = 35 and 44 μm, respectively). Upon expression in Xenopus laevis oocytes it was similarly observed that Zn2+ was a potent inhibitor of the GABA-induced current (IC50 = 21 μm for T349H/E370H and 51 μm for T349H/Q374C), albeit maximum inhibition was only ∼40% in T349H/E370H versus ∼90% in T349H/Q374C. In the wild type, Zn2+ did not affect the Na+-dependent transient currents elicited by voltage jumps and thought to reflect capacitive charge movements associated with Na+ binding. However, in both mutants Zn2+ caused a reduction of the inward transient currents upon jumping to hyperpolarized potentials as reflected in rightward-shifted Q/V relationships. This suggests that Zn2+ is inhibiting transporter function by stabilizing the outward-facing Na+-bound state. Translocation of lithium by the transporter does not require GABA binding and analysis of this uncoupled Li+ conductance revealed a potent inhibition by Zn2+ in T349H/E370H, whereas surprisingly the T349H/Q374C leak was unaffected. This differential effect supports that the leak conductance represents a unique operational mode of the transporter involving conformational changes different from those of the substrate translocation process. Altogether our results support both an evolutionary conserved structural organization of the TM 7/8 domain and a key role of this domain in GABA-dependent and -independent conformational changes of the transporter.