Aquaporin‐4 regulates the velocity and frequency of cortical spreading depression in mice

The astrocyte water channel aquaporin‐4 (AQP4) regulates extracellular space (ECS) K⁺ concentration ([K⁺]_e) and volume dynamics following neuronal activation. Here, we investigated how AQP4‐mediated changes in [K⁺]_e and ECS volume affect the velocity, frequency, and amplitude of cortical spreading depression (CSD) depolarizations produced by surface KCl application in wild‐type (AQP4^+/+) and AQP4‐deficient (AQP4^?/?) mice. In contrast to initial expectations, both the velocity and the frequency of CSD were significantly reduced in AQP4^?/? mice when compared with AQP4^+/+ mice, by 22% and 32%, respectively. Measurement of [K⁺]_e with K⁺‐selective microelectrodes demonstrated an increase to ～35 mM during spreading depolarizations in both AQP4^+/+ and AQP4^?/? mice, but the rates of [K⁺]_e increase (3.5 vs. 1.5 mM/s) and reuptake (t_1/2 33 vs. 61 s) were significantly reduced in AQP4^?/? mice. ECS volume fraction measured by tetramethylammonium iontophoresis was greatly reduced during depolarizations from 0.18 to 0.053 in AQP4^+/+ mice, and 0.23 to 0.063 in AQP4^?/? mice. Analysis of the experimental data using a mathematical model of CSD propagation suggested that the reduced velocity of CSD depolarizations in AQP4^?/? mice was primarily a consequence of the slowed increase in [K⁺]_e during neuronal depolarization. These results demonstrate that AQP4 effects on [K⁺]_e and ECS volume dynamics accelerate CSD propagation. GLIA 2015;63:1860–1869     

Principles of sodium homeostasis and sodium signalling in astroglia

Sodium homeostasis is at the center stage of astrocyte (and brain) physiology because the large inwardly directed Na⁺ gradient provides the energy for transport of ions, neurotransmitters, amino acids and many other molecules across the plasmalemma and endomembranes. Cell imaging with commercially available chemical indicators allows analysis of dynamic changes in intracellular Na⁺ concentration (Na⁺]_i), albeit further technological developments, such as genetically‐controlled or membrane targeted indicators or dyes usable for advanced microscopy (such as fluorescence‐lifetime imaging microscopy) are urgently needed. Thus, important questions related to the existence of Na⁺ gradients between different cellular compartments or occurrence of localised Na⁺ micro/nanodomains at the plasma membrane remain debatable. Extrusion of Na⁺ (and hence Na⁺ homeostasis) in astrocytes is mediated by the ubiquitously expressed Na⁺/K⁺‐ATPase (NKA), the major energy consumer of the brain. The activity of the NKA is counteracted by constant constitutive influx of Na⁺ through transporters such as the NKCC1 (Na⁺‐K⁺?2Cl^‐‐co‐transport) or the NBC (Na⁺?2 ‐co‐transport). In addition, Na⁺‐permeable ion channels at the plasma membrane as well as Na⁺‐dependent solute carrier transporters provide for Na⁺ influx into astrocytes. Activation of these pathways in response to neuronal activity results in an increase of [Na⁺]_i in astrocytes and there is manifold evidence for diverse signalling functions of these [Na⁺]_i transients. Thus, in addition to its established homeostatic functions, activity dependent fluctuations of astrocyte [Na⁺]_i regulate signalling cascades by feeding back on Na⁺‐dependent transporters. The Na⁺ signalling system may be ideally placed for fast coordinating signalling between neuronal activity and glial “homeostatic” Na⁺‐dependent transporters. GLIA 2016;64:1611–1627     

Involvement of vH+‐ATPase in synaptic vesicle swelling

Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G_αi3‐PLA2‐mediated involvement of water channel AQP‐1 in the regulation of secretory vesicle swelling in exocrine pancreas and the G_αo‐mediated AQP‐6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH⁺‐ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer‐scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH⁺‐ATPase in GTP‐G_αo‐mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP‐G_αo‐mediated vesicle swelling is vH⁺‐ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin‐sensitive vesicle acidification, following the GTP‐G_αo‐induced swelling stimulus. Water channels are bidirectional and the vH⁺‐ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP‐mastoparan stimulated swelling, suggesting that vH⁺‐ATPase is upstream of AQP‐6, in the pathway leading from G_αo‐stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP‐6‐mediated gating of water into synaptic vesicles. © 2009 Wiley‐Liss, Inc.     

Clearance of activity‐evoked K+ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform‐selective AQP4 inhibitor

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4‐deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4^?/? mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4‐expressing Xenopus laevis oocytes monitored by a high‐resolution volume recording system, we here demonstrate that the compound TGN‐020 is such a selective AQP4 inhibitor. TGN‐020 targets the tested species of AQP4 with an IC₅₀ of ~3.5 μM, but displays no inhibitory effect on the other AQPs (AQP1‐AQP9). With this tool, we employed rat hippocampal slices and ion‐sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN‐020‐mediated inhibition of AQP4 did not prevent stimulus‐induced extracellular space shrinkage, nor did it slow clearance of the activity‐evoked K⁺ transient. These data, obtained with a verified isoform‐selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K⁺ clearance or the associated extracellular space shrinkage.     

Molecular dissection of the myelinated axon

The membrane of the myelinated axon expresses a rich repertoire of physiologically active molecules: (1) Voltagesensitive NA⁺ channels are clustered at high density (～1,000/μm²) in the nodal axon membrane and are present at lower density(<25/μm²) in the internodal axon membrane under the myelin. Na⁺ channels are also present within Schwann cell processes (in peripheral nerve) and perinodal astrocyte processes (in the central nervous system) which contact the Na⁺ channel–rich axon membrane at the node. In some demyelinated fibers, the bared (formerly internodal) axon membrane reorganizes and expresses a higher-than-normal Na⁺ channel density, providing a basis for restoration of conduction. The presence of glial cell processes, adjacent to foci of Na⁺ channels in immature and demyelinated axons, suggests that glial cells participate in the clustering of Na⁺ channels in the axon membrane. (2) “Fast” K⁺ channels, sensitive to 4-aminopyridine, are present in the paranodal of internodal axon membrane under the myelin; these channels may function to prevent reexcitation following action potentials, or participate in the generation of an internodal resting potential. (3) “Slow” K⁺ channels, sensitive to tetraethylammonium, are present in the nodal axon membrane and, in lower densities, in the internodal axon membrane; their activation produces a hyperpolarizing afterpotential which modulates repetitive firing. (4) The “inward rectifier” is activated by hyperpolarization. This channel is permeable to both Na⁺ and K⁺ ions and may modulate axonal excitability or participate in ionic reuptake following activity. (5) Na⁺/K⁺-ATPase and (6) Ca²⁺-ATPase are also present in the axon membrane and function to maintain transmembrane gradients of Na⁺, K⁺, and Ca²⁺. (7) A specialized antiporter molecule, the Na⁺/Ca²⁺ exchanger, is present in myelinated axons within central nervous system white matter. Following anoxia, the Na⁺/Ca²⁺ exchanger mediates an influx of Ca²⁺ which damages the axon. The molecular organization of the myelinated axon has important pathophysiological implications. Blockade of fast K⁺ channels and Na⁺/K⁺-ATPase improves action potential conduction in some demyelinated axons, and block of the Na⁺/Ca²⁺ exchanger protects white matter axons from anoxic injury. Modification of ion channels, pumps, and exchangers in myelinated fibers may thus provide an important therapeutic approach for a number of neurological disorders.     

The role of aquaporin‐4 and transient receptor potential vaniloid isoform 4 channels in the development of cytotoxic edema and associated extracellular diffusion parameter changes

The proper function of the nervous system is dependent on the balance of ions and water between the intracellular and extracellular space (ECS). It has been suggested that the interaction of aquaporin‐4 (AQP4) and the transient receptor potential vaniloid isoform 4 (TRPV4) channels play a role in water balance and cell volume regulation, and indirectly, of the ECS volume. Using the real‐time iontophoretic method, we studied the changes of the ECS diffusion parameters: ECS volume fraction α (α = ECS volume fraction/total tissue volume) and tortuosity λ (λ² = free/apparent diffusion coefficient) in mice with a genetic deficiency of AQP4 or TRPV4 channels, and in control animals. The used models of cytotoxic edema included: mild and severe hypotonic stress or oxygen‐glucose deprivation (OGD) in situ and terminal ischemia/anoxia in vivo. This study shows that an AQP4 or TRPV4 deficit slows down the ECS volume shrinkage during severe ischemia in vivo. We further demonstrate that a TRPV4 deficit slows down the velocity and attenuates an extent of the ECS volume decrease during OGD treatment in situ. However, in any of the cytotoxic edema models in situ (OGD, mild or severe hypotonic stress), we did not detect any alterations in the cell swelling or volume regulation caused by AQP4 deficiency. Overall, our results indicate that the AQP4 and TRPV4 channels may play a crucial role in severe pathological states associated with their overexpression and enhanced cell swelling. However, detailed interplay between AQP4 and TRPV4 channels requires further studies and additional research.     

Changes in the astrocytic aquaporin‐4 and inwardly rectifying potassium channel expression in the brain of the amyotrophic lateral sclerosis SOD1G93A rat model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood–brain barrier (BBB), are enriched in two proteins, aquaporin‐4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1^G93A rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole‐cell patch‐clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir‐specific Cs⁺‐ and Ba²⁺‐sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death. © 2012 Wiley Periodicals, Inc.     

KATP channel openers protect mesencephalic neurons against MPP+‐induced cytotoxicity via inhibition of ROS production

Opening of ATP‐sensitive potassium (K_ATP) channels has been demonstrated to exert significant neuroprotection in in vivo and in vitro models of Parkinson's disease (PD), but the exact mechanism remains unclear. In the present study, various K_ATP channel openers (KCOs) sensitive to diverse K_ATP subunits were used to clarify the protective role of K_ATP channel opening in 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced oxidative stress injury in mouse primary cultured mesencephalic neurons. The results showed that pretreatment with nonselective KCO pinacidil (Pin) or diazoxide (Dia), a KCO sensitive to Kir6.2/SUR1 K_ATP channels, protected mesencephalic neurons, especially dopaminergic neurons, against MPP⁺‐induced injury in a concentration‐dependent manner. However, cromakalim (Cro), an opener of Kir6.1/SUR2 but not Kir6.2/SUR1 K_ATP channels, failed to protect against MPP⁺‐induced cytotoxicity. Furthermore, Pin and Dia but not Cro significantly suppressed the elevation of reactive oxygen species (ROS) triggered by MPP⁺ and prevented the loss of mitochondrial member potential (ΔΨm) and the release of mitochondrial cyotchrome c. Consequently, opening of K_ATP channels expressed in neurons could protect primary mesencephalic neurons against MPP⁺‐induced cytotoxicity via inhibiting ROS overproduction and subsequently ameliorating mitochondrial function. © 2009 Wiley‐Liss, Inc.     

Aquaporin‐4 in manganese‐treated cultured astrocytes

Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin‐4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen‐activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal‐regulated kinase (ERK)1/2, C‐Jun amino‐terminal kinase (JNK)1/2/3, and p38‐MAPK. Inhibitors of ERK1/2 and p38‐MAPK (but not of JNK) blocked (40–60%) the manganese‐induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4‐related neurological complications associated with manganese toxicity. © 2010 Wiley‐Liss, Inc.     

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.     

Iptakalim ameliorates MPP+‐induced astrocyte mitochondrial dysfunction by increasing mitochondrial complex activity besides opening mitoKATP channels

In addition to the established role of the mitochondrion in energy metabolism, regulation of cell death has been regarded as a major function of this organelle. Our previous studies have demonstrated that iptakalim (IPT), a novel ATP‐sensitive potassium channel (K_ATP channel) opener, protects against 1‐methyl‐4‐phenyl‐pyridinium ion (MPP⁺)–induced astrocyte apoptosis via mitochondria and mitogen‐activated protein kinase signal pathways. The present study aimed to investigate whether IPT can protect astrocyte mitochondria against MPP⁺‐induced mitochondrial dysfunction. We showed that treatment with IPT could ameliorate the inhibitory effect of MPP⁺ on mitochondrial respiration and ATP production by using mitochondrial complex I–supported substrates. IPT could also inhibit the increased production of mitochondrial reactive oxygen species (ROS) and the release of cytochrome c from mitochondria induced by MPP⁺. However, mitochondrial ATP‐sensitive potassium (mitoK_ATP) channel blocker 5‐hydroxydecanoate (5‐HD) could partly abolish all of the above effects of IPT. Because mitochondrial complex dysfunction impairs mitochondrial respiration and ATP production, a further experiment was undertaken to study the effects of IPT on the activity of mitochondrial complex (COX) I and COX IV. It was found that IPT inhibited the decrease in mitochondrial COX I and COX IV activity induced by MPP⁺, but 5‐HD failed to abolish these effects. Taken together, these findings suggest that IPT may protect astrocyte mitochondrial function by regulating complex activity in addition to opening mitoK_ATP channels. © 2008 Wiley‐Liss, Inc.     

AQP5 is differentially regulated in astrocytes during metabolic and traumatic injuries

Water movement plays vital roles in both physiological and pathological conditions in the brain. Astrocytes are responsible for regulating this water movement and are the major contributors to brain edema in pathological conditions. Aquaporins (AQPs) in astrocytes play critical roles in the regulation of water movement in the brain. AQP1, 3, 4, 5, 8, and 9 have been reported in the brain. Compared with AQP1, 4, and 9, AQP3, 5, and 8 are less studied. Among the lesser known AQPs, AQP5, which has multiple functions identified outside the central nervous system, is also indicated to be involved in hypoxia injury in astrocytes. In our study, AQP5 expression could be detected both in primary cultures of astrocytes and neurons, and AQP5 expression in astrocytes was confirmed in 1‐ to 4‐week old primary cultures of astrocytes. AQP5 was localized on the cytoplasmic membrane and in the cytoplasm of astrocytes. AQP5 expression was downregulated during ischemia treatment and upregulated after scratch‐wound injury, which was also confirmed in a middle cerebral artery occlusion model and a stab‐wound injury model in vivo. The AQP5 increased after scratch injury was polarized to the migrating processes and cytoplasmic membrane of astrocytes in the leading edge of the scratch‐wound, and AQP5 over‐expression facilitated astrocyte process elongation after scratch injury. Taken together, these results indicate that AQP5 might be an important water channel in astrocytes that is differentially expressed during various brain injuries. GLIA 2013;61:1748–1765     

Role of TRPM2 cation channels in dorsal root ganglion of rats after experimental spinal cord injury

Introduction: We sought to determine the contribution of oxidative stress–dependent activation of TRPM2 and L‐type voltage‐gated Ca²⁺ channels (VGCC) in dorsal root ganglion (DRG) neurons of rats after spinal cord injury (SCI). Methods: The rats were divided into 4 groups: control; sham control; SCI; and SCI+nimodipine groups. The neurons of the SCI groups were also incubated with non‐specific TRPM2 channel blockers, 2‐aminoethoxydiphenylborate (2‐APB) and N‐(p‐amylcinnamoyl)anthranilic acid (ACA), before H₂O₂ stimulation. Results:The [Ca²⁺]_i concentrations were higher in the SCI group than in the control groups, although their concentrations were decreased by nimodipine and 2‐APB. The H₂O₂‐induced TRPM2 current densities in patch‐clamp experiments were decreased by ACA and 2‐APB incubation. In the nimodipine group, the TRPM2 channels of neurons were not activated by H₂O₂ or cumene hydroperoxide. Conclusions: Increased Ca²⁺ influx and currents in DRG neurons after spinal injury indicated TRPM2 and voltage‐gated Ca²⁺ channel activation. Muscle Nerve 48 : 945–950, 2013     

Phosphorylation of rat aquaporin‐4 at Ser111 is not required for channel gating

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 Ser¹¹¹ 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 Ser¹¹¹ 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 Ser¹¹¹ 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.Ser¹¹¹ recorded no phosphorylation‐induced change in water permeability. A phospho‐specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser¹¹¹, 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 Ser¹¹¹ and of phosphorylation‐dependent gating of AQP4.     

Astrocyte‐mediated infantile‐onset leukoencephalopathy mouse model

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile‐onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte‐specific Mlc1 overexpression causes infantile‐onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na⁺/K⁺ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na⁺/K⁺ pump. In contrast, no changes in Na⁺/K⁺ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na⁺/K⁺ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile‐onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile‐onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150–168     

Activation and modulation of calcium-activated non-selective cation channels from embryonic chick sensory neurons

We have shown that calcium-activated non-selective (CAN) channels from embryonic chick sensory neurons are permeable to both Na⁺ and K⁺ and are not blocked by TTX, TEA, or 4-AP. These neuronal CAN channels are activated by sub-micromolar cytoplasmic Ca²⁺ with negative cooperativity. The effect of Ca²⁺ is to decrease the closed times of the channel with little effect on the time the channel remains open. Isolated neuronal CAN channels can be phosphorylated by cAMP-dependent protein kinase (PKA). The effect of phosphorylation is to shorten channel open time and to minimize the effect of Ca²⁺ on channel closed time.     

High sensitivity of spontaneous spike frequency to sodium leak current in a Lymnaea pacemaker neuron

The spontaneous rhythmic firing of action potentials in pacemaker neurons depends on the biophysical properties of voltage‐gated ion channels and background leak currents. The background leak current includes a large K⁺ and a small Na⁺ component. We previously reported that a Na⁺‐leak current via U‐type channels is required to generate spontaneous action potential firing in the identified respiratory pacemaker neuron, RPeD1, in the freshwater pond snail Lymnaea stagnalis. We further investigated the functional significance of the background Na⁺ current in rhythmic spiking of RPeD1 neurons. Whole‐cell patch‐clamp recording and computational modeling approaches were carried out in isolated RPeD1 neurons. The whole‐cell current of the major ion channel components in RPeD1 neurons were characterized, and a conductance‐based computational model of the rhythmic pacemaker activity was simulated with the experimental measurements. We found that the spiking rate is more sensitive to changes in the Na⁺ leak current as compared to the K⁺ leak current, suggesting a robust function of Na⁺ leak current in regulating spontaneous neuronal firing activity. Our study provides new insight into our current understanding of the role of Na⁺ leak current in intrinsic properties of pacemaker neurons.     

AQP4 Knockout Aggravates Ischemia/Reperfusion Injury in Mice

SUMMARY Background and Purpose: The glial water channel aquaporin‐4 (AQP4) has been shown to be involved in a wide range of brain disorders. Although its important role in stroke has already been documented, the underlying mechanism was not clarified yet. Therefore, this study was designed to investigate the impacts of AQP4 deletion in ischemia/reperfusion (I/R). Methods and Results: Herein we found a higher mortality and more severe neurological deficits in AQP4 knockout (AQP4^?/?) mice after transient middle cerebral artery occlusion while no difference was observed in water content variation during I/R between two genotypes except a higher basal water content developed in AQP4^?/? mouse brain, implying the same increment of water content over a higher basal level may provoke an even more elevated intracranial pressure, which might be an important cause of increased mortality in AQP4^?/? mice. Moreover, AQP4 knockout aggravated I/R injury with enlarged infarct size and a more serious loss of CA1 neurons accompanied by a striking hypertrophy of astrocytes, suggesting an involvement of AQP4 in astrocytic dysfunction. Conclusions: Our findings provide direct evidence that AQP4 plays a crucial role in the pathogenesis of I/R injury, which may confer a new option for stroke treatment.