Hana Inoue

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress

Astrocytes release glutamate upon hyperexcitation in the normal brain, and in response to pathologic insults such as ischemia and trauma. In our experiments, both hypotonic and ischemic stimuli caused the release of glutamate from cultured mouse astrocytes, which occurred with little or no contribution of gap junction hemichannels, vesicle‐mediated exocytosis, or reversed operation of the Na‐dependent glutamate transporter. Cell swelling and chemical ischemia activated, in cell‐attached membrane patches, anionic channels with large unitary conductance (∼400 pS) and inactivation kinetics at potentials more positive than +20 mV or more negative than −20 mV. These properties are different from those of volume‐sensitive outwardly rectifying (VSOR) Cl− channels, which were also expressed in these cells and exhibited intermediate unitary conductance (∼80 pS) and inactivation kinetics at large positive potentials of more than +40 mV. Both maxi‐anion channels and VSOR Cl− channels were permeable to glutamate with permeability ratios of glutamate to chloride of 0.21 ± 0.07 and 0.15 ± 0.01, respectively. However, the release of glutamate was significantly more sensitive to Gd3+, a blocker of maxi‐anion channels, than to phloretin, a blocker of VSOR Cl− channels. We conclude that these two channels jointly represent a major conductive pathway for the release of glutamate from swollen and ischemia‐challenged astrocytes, with the contribution of maxi‐anion channels being predominant.

Roles of Volume-Sensitive Chloride Channel in Excitotoxic Neuronal Injury

Excitotoxicity is associated with stroke, brain trauma, and a number of neurodegenerative disorders. In the brain, during excitotoxic insults, neurons undergo rapid swelling in both the soma and dendrites. Focal swellings along the dendrites called varicosities are considered to be a hallmark of acute excitotoxic neuronal injury. However, it is not clear what pathway is involved in the neuronal anion flux that leads to the formation and resolution of excitotoxic varicosities. Here, we assessed the roles of the volume-sensitive outwardly rectifying (VSOR) Cl− channel in excitotoxic responses in mouse cortical neurons. Whole-cell patch-clamp recordings revealed that the VSOR Cl− channel in cultured neurons was activated by NMDA exposure. Moreover, robust expression of this channel on varicosities was confirmed by on-cell and nystatin-perforated vesicle patch techniques. VSOR channel blockers, but not blockers of GABAA receptors and Cl− transporters, abolished not only varicosity resolution after sublethal excitotoxic stimulation but also necrotic death after sustained varicosity formation induced by prolonged NMDA exposure in cortical neurons. The present slice-patch experiments demonstrated, for the first time, expression of the VSOR Cl− channels in somatosensory pyramidal neurons. NMDA-induced necrotic neuronal death in slice preparations was largely suppressed by a blocker of the VSOR Cl− channel but not of the GABAA receptor. These results indicate that VSOR Cl− channels exert dual, reciprocal actions on neuronal excitotoxicity by serving as major anionic pathways both for varicosity recovery after washout of an excitotoxic stimulant and for persistent varicosity formation under prolonged excitotoxic insults leading to necrosis in cortical neurons.

Volume-sensitive chloride channels in mouse cortical neurons: characterization and role in volume regulation

Because persistent swelling causes cell damage and often results in cell death, volume regulation is an important physiological function in both neuronal and non‐neuronal cells. Brain cell swelling has been observed not only in various pathological conditions but also during physiological synaptic transmissions. Volume‐sensitive anion channels have been reported to play an important role in the regulatory volume decrease occurring after osmotic swelling in many cell types. In this study, using a two‐photon laser scanning microscope and patch‐clamp techniques, we found that mouse cortical neurons in primary culture exhibit regulatory volume decrease after transient swelling and activation of Cl– currents during exposure to a hypotonic solution. The regulatory volume decrease was inhibited by Cl– channel blockers or K+ channel blockers. Swelling‐activated Cl– currents exhibited outward rectification, time‐dependent inactivation at large positive potentials, a low‐field anion permeability sequence, an intermediate unitary conductance and sensitivity to known blockers of volume‐sensitive Cl– channels. Thus, it is concluded that the activity of the volume‐sensitive outwardly rectifying Cl– channel plays a role in the control of cell volume in cortical neurons.

Roles of Volume-sensitive Cl− Channel in Cisplatin-induced Apoptosis in Human Epidermoid Cancer Cells

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl−/HCO3− exchangers and Cl− channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO3−, we investigated the relation between cisplatin-induced apoptosis and Cl− channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl− channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl− current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl− channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.

Anion channel blockers attenuate delayed neuronal cell death induced by transient forebrain ischemia

Chloride efflux is known to be involved in the progression of apoptosis in various cell types. We have recently shown that the volume‐sensitive outwardly rectifying (VSOR) anion channel serves as the pathway for apoptotic chloride efflux in some cells. In the present study, we tested the neuroprotective effects of drugs that can block the VSOR anion channel, on delayed neuronal death (DND) induced by transient forebrain ischemia. The functional expression of the VSOR anion channel was first examined in hippocampal neurons in both primary culture and hippocampal slice preparations, by the whole‐cell patch‐clamp technique. We then tested the channels sensitivity to an anion channel blocker, 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), and a tyrosine kinase blocker, genistein. By histological examinations and cytochrome c release assessments, the protective effects of these drugs on the DND of hippocampal CA1 neurons in mice subjected to transient ischemia were examined. Drugs were administered via the jugular vein prior to ischemic treatment and into the peritoneal cavity after reperfusion. Hippocampal neurons were found to express the volume‐sensitive Cl– channel, which exhibits outward rectification and is sensitive to DIDS and genistein. Administration of DIDS or genistein reduced cytochrome c release and the number of damaged neurons in the CA1 region after transient forebrain ischemia. This fact suggests that the DND induction mechanism involves the activity of the VSOR anion channel and that this channel may provide a therapeutic target for the treatment of stroke.

Mutation of the Mg2+ Transporter SLC41A1 Results in a Nephronophthisis-Like Phenotype

Nephronophthisis (NPHP)-related ciliopathies are recessive, single-gene disorders that collectively make up the most common genetic cause of CKD in the first three decades of life. Mutations in 1 of the 15 known NPHP genes explain less than half of all cases with this phenotype, however, and the recently identified genetic causes are exceedingly rare. As a result, a strategy to identify single-gene causes of NPHP-related ciliopathies in single affected families is needed. Although whole-exome resequencing facilitates the identification of disease genes, the large number of detected genetic variants hampers its use. Here, we overcome this limitation by combining homozygosity mapping with whole-exome resequencing in a sibling pair with an NPHP-related ciliopathy. Whole-exome capture revealed a homozygous splice acceptor site mutation (c.698G>T) in the renal Mg(2+) transporter SLC41A1. This mutation resulted in skipping of exon 6 of SLC41A1, resulting in an in-frame deletion of a transmembrane helix. Transfection of cells with wild-type or mutant SLC41A1 revealed that deletion of exon 6 completely blocks the Mg(2+) transport function of SLC41A1. Furthermore, in normal human kidney tissue, endogenous SLC41A1 specifically localized to renal tubules situated at the corticomedullary boundary, consistent with the region of cystogenesis observed in NPHP and related ciliopathies. Last, morpholino-mediated knockdown of slc41a1 expression in zebrafish resulted in ventral body curvature, hydrocephalus, and cystic kidneys, similar to the effects of knocking down other NPHP genes. Taken together, these data suggest that defects in the maintenance of renal Mg(2+) homeostasis may lead to tubular defects that result in a phenotype similar to NPHP.

The Puzzles of Volume-Activated Anion Channels

This chapter deals with the anion channels and their multiple functions. Anion channels (ACs) are present both in the plasma membrane and in the membranes of intracellular organelles. Animal cells express a large variety of anion channels in their plasma membrane. ACs are involved in a wide range of functions, such as inhibitory synaptic transmission through plasma membrane hyperpolarization, epithelial Clˉ transport, as well as transport of other organic anions such as glutamate and anionic forms of ATP. In contrast to cation channels, ACs are not directly involved in the initiation and termination of action potentials in nerves and muscles. In neurons and other cell types, membrane potential is determined by the relative electromotive forces and the conductances of each ion permeation pathway.

Volume-sensitive outwardly rectifying chloride channel in white adipocytes from normal and diabetic mice

The volume-sensitive outwardly rectifying (VSOR) chloride channel is ubiquitously expressed and involved in cell volume regulation after osmotic swelling, called regulatory volume decrease (RVD), in various cell types. In adipocytes, the expression of the VSOR channel has not been explored to date. Here, by employing the whole-cell patch-clamp technique, we examined whether or not the VSOR channel is expressed in white adipocytes freshly isolated from epididymal fat pads of normal (C57BL/6 or KK) and diabetic (KKA(y)) mice. Whole cell voltage-clamp recordings revealed that Cl(-) currents were gradually activated upon cell swelling induced by application of a hypotonic solution, both in normal and diabetic adipocytes. Although both the mean cell size (or cell capacitance) and the current magnitude in KKA(y) adipocytes were larger than those in C57BL/6 cells, the current density was significantly lower in KKA(y) adipocytes (23.32 +/- 1.94 pA in C57BL/6 adipocytes vs. 13.04 +/- 2.41 pA in KKA(y) adipocytes at +100 mV). Similarly, the current density in diabetic KKA(y) adipocytes was lower than that in adipocytes from KK mice (a parental strain of KKA(y) mice), which do not present diabetes until an older age. The current was inhibited by Cl(-) channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and glibenclamide, or hypertonic solution, and showed outward rectification and inactivation kinetics at large positive potentials. These electrophysiological and pharmacological properties are consistent with those of the VSOR channel in other cell types. Moreover, adipocytes showed RVD, which was inhibited by NPPB. In KKA(y) adipocytes, RVD was significantly slower (tau; 8.42 min in C57BL/6 adipocytes vs. 11.97 min in KKA(y) adipocytes) and incomplete during the recording period (25 min). It is concluded that the VSOR channel is functionally expressed and involved in volume regulation in white adipocytes. RVD is largely impaired in adipocytes from diabetic mice, presumably as a consequence of the lower density of the functional VSOR channel in the plasma membrane.

Expression of novel isoforms of the CIC-1 chloride channel in astrocytic glial cells in vitro

Chloride channels play an important role in glial astrocyte function. However, in astrocytes, no chloride channels besides the γ‐aminobutyric acid (GABA)A receptor, glycine receptor, and ClC‐2 chloride channels have been molecularly identified. In this study, we examined the expression of the ClC‐1 chloride channel in rat astrocytic glioma C6 cells and rat primary astrocytes. Five isoforms of ClC‐1, but not skeletal muscle ClC‐1 (SM ClC‐1), were found to be expressed in C6 cells. Comparison with rat SM ClC‐1 showed that common features shared by these isoforms are a short 3′ end with a deletion of the nucleotides from 3115 to 3197 and a substitution of T by C at nucleotides 480 and 1733. Three of the five isoforms, M1, M2, and M3, were produced by partial deletion of ClC‐1 exon 7, partial insertion of ClC‐1 exon 7a, and a TAG insertion at nucleotide 858, respectively. One of the two remaining isoforms, M4, was produced by partial deletion of ClC‐1 exon 8 at nucleotide 937; the other, M5, was the same as SM ClC‐1 except for the short 3′ end and substitutions at the two positions. Only the M5 isoform could be expressed as a functional channel in Xenopus oocytes. This glial isoform exhibited less dependence on voltage and extracellular Cl− than rat SM ClC‐1. However, the anion selectivity sequence and the anthracene‐9‐carboxylic acid (9‐AC) sensitivity of this channel were the same as for SM ClC‐1. Since whole‐cell recordings failed to detect ClC‐1‐like Cl− currents in C6 cells, it appears that the ClC‐1 isoform is functioning in intracellular organelles. In rat primary astrocytes, we found that the M2 isoform as well as two additional distinct isoforms were expressed. The present study showed that astrocytic glial cells express multiple isoforms of the ClC‐1 chloride channel, which has been thought to be expressed almost exclusively in the skeletal muscle.

Magnesium Homeostasis in Cardiac Myocytes of Mg-Deficient Rats

To study possible modulation of Mg2+ transport in low Mg2+ conditions, we fed either a Mg-deficient diet or a Mg-containing diet (control) to Wistar rats for 1–6 weeks. Total Mg concentrations in serum and cardiac ventricular tissues were measured by atomic absorption spectroscopy. Intracellular free Mg2+ concentration ([Mg2+]i) of ventricular myocytes was measured with the fluorescent indicator furaptra. Mg2+ transport rates, rates of Mg2+ influx and Mg2+ efflux, were estimated from the rates of change in [Mg2+]i during Mg loading/depletion and recovery procedures. In Mg-deficient rats, the serum total Mg concentration (0.29±0.026 mM) was significantly lower than in control rats (0.86±0.072 mM) after 4–6 weeks of Mg deficiency. However, neither total Mg concentration in ventricular tissues nor [Mg2+]i of ventricular myocytes was significantly different between Mg-deficient rats and control rats. The rates of Mg2+ influx and efflux were not significantly different in both groups. In addition, quantitative RT-PCR revealed that Mg deficiency did not substantially change mRNA expression levels of known Mg2+ channels/transporters (TRPM6, TRPM7, MagT1, SLC41A1 and ACDP2) in heart and kidney tissues. These results suggest that [Mg2+]i as well as the total Mg content of cardiac myocytes, was well maintained even under chronic hypomagnesemia without persistent modulation in function and expression of major Mg2+ channels/transporters in the heart.