A proton channel, Otopetrin 1 (OTOP1) is N-glycosylated at two asparagine residues in third extracellular loop.

A proton (H+) channel, Otopetrin 1 (OTOP1) is an acid sensor in the sour taste receptor cells. Although OTOP1 is known to be activated by extracellular acid, no posttranslational modification of OTOP1 has been reported. As one of the posttranslational modifications, glycosylation is known to modulate many ion channels. In this study, we investigated whether OTOP1 is glycosylated and how the glycosylation affects OTOP1 function. Pharmacological and enzymatic examinations (using an N-glycosylation inhibitor, tunicamycin and peptide: N-glycanase F [PNGase F]) revealed that overexpressed mouse OTOP1 was N-glycosylated. As the N-glycans were Endoglycosidase H (Endo H)-sensitive, they were most likely high-mannose type. A site-directed mutagenesis approach revealed that both two asparagine residues (N238 and N251) in the third extracellular loop between the fifth transmembrane region and the sixth transmembrane region (L5-6) were the glycosylation sites. Prevention of the glycosylations by the mutations of the asparagine residues or by tunicamycin treatment diminished the whole-cell OTOP1 current densities. The results of cell surface biotinylation assay showed that the prevention of the glycosylations reduced the surface expression of OTOP1 at the plasma membrane. These results indicate that mouse OTOP1 is N-glycosylated at N238 and N251, and that the glycosylations are necessary for OTOP1 to show the maximum degree of H+ current densities at the plasma membrane through promoting its targeting to the plasma membrane. These findings on glycosylations of OTOP1 will be a part of a comprehensive understanding on the regulations of OTOP1 function.

Cibacron blue 3G-A is a novel inhibitor of Otopetrin 1 (OTOP1), a proton channel.

Otopetrin 1 (OTOP1) is a proton (H+) channel which detects acidic stimuli in sour taste receptor cells and plays some sort of role in the formation of otoconia in the inner ear. Although it is known that zinc ion (Zn2+) inhibits OTOP1, Zn2+ requires high concentrations (mM order) to inhibit OTOP1 sufficiently, and no other inhibitors have been found. Therefore, to identify a novel inhibitor, we screened a chemical library (LOPAC1280) by whole-cell patch clamp recordings, measuring proton currents of heterologously-expressed mouse OTOP1. From the screening, we found that reactive blue 2 inhibited OTOP1 currents. Further evaluations of three analogues of reactive blue 2 revealed that cibacron blue 3G-A potently inhibited OTOP1 currents. Cibacron blue 3G-A inhibited OTOP1 currents in a concentration-dependent manner, and its 50% inhibitory concentration (IC50) and the Hill coefficient were 5.0 µM and 1.1, respectively. The inhibition of OTOP1 currents by cibacron blue 3G-A was less affected by extracellular anion compositions, membrane potentials, and low pH than the inhibition by Zn2+. These results suggest that the inhibition of OTOP1 by cibacron blue 3G-A is neither likely to be a pore-blocking inhibition nor a competitive inhibition. Furthermore, our findings revealed that cibacron blue 3G-A can be used as a novel inhibitor of OTOP1 especially under the conditions in which OTOP1 activity is evaluated such as low pH.

Single-Molecule Conductance of a π-Hybridized Tripodal Anchor while Maintaining Electronic Communication.

Direct hybridization between the π-orbital of a conjugated molecule and metal electrodes is recognized as a new anchoring strategy to enhance the electrical conductance of single-molecule junctions. The anchor is expected to maintain direct hybridization between the conjugated molecule and the metal electrodes, and control the orientation of the molecule against the metal electrodes. However, fulfilling both requirements is difficult because multipodal anchors aiming at a robust contact with the electrodes often break the π-conjugation, thereby resulting in an inefficient carrier transport. Herein, a new tripodal anchor framework-a 7,7-diphenyl-7H-benzo[6,7]indeno[1,2-b]thiophene (PBIT) derivative-is developed. In this framework, π-conjugation is maintained in the molecular junction, and the tripodal structure makes the molecule stand upright on the metal electrode. Molecular conductance is measured by the break junction technique. A vector-based classification and first-principles transport calculations determine the single-molecule conductance of the tripodal-anchoring structure. The conductance of the PBIT-based molecule is higher than that of the tripodal anchor having sp3 carbon atoms in the carrier transport pathway. These results demonstrate that extending the π-conjugation to the tripodal leg is an effective strategy for enhancing the conductivities of single-molecule junctions.

Opposing functions of α- and ß-adrenoceptors in the formation of processes by cultured astrocytes.

Astrocytes are glial cells with numerous fine processes which are important for the functions of the central nervous system. The activation of ß-adrenoceptors induces process formation of astrocytes via cyclic AMP (cAMP) signaling. However, the role of α-adrenoceptors in the astrocyte morphology has not been elucidated. Here, we examined it by using cultured astrocytes from neonatal rat spinal cords and cortices. Exposure of these cells to noradrenaline and the ß-adrenoceptor agonist isoproterenol increased intracellular cAMP levels and induced the formation of processes. Noradrenaline-induced process formation was enhanced with the α1-adrenoceptor antagonist prazosin and α2-adrenoceptor antagonist atipamezole. Atipamezole also enhanced noradrenaline-induced cAMP elevation. Isoproterenol-induced process formation was not inhibited by the α1-adrenoceptor agonist phenylephrine but was inhibited by the α2-adrenoceptor agonist dexmedetomidine. Dexmedetomidine also inhibited process formation induced by the adenylate cyclase activator forskolin and the membrane-permeable cAMP analog dibutyryl-cAMP. Moreover, dexmedetomidine inhibited cAMP-independent process formation induced by adenosine or the Rho-associated kinase inhibitor Y27632. In the presence of propranolol, noradrenaline inhibited Y27632-induced process formation, which was abolished by prazosin or atipamezole. These results demonstrate that α-adrenoceptors inhibit both cAMP-dependent and -independent astrocytic process formation.

A Mechanosensitive Channel, Mouse Transmembrane Channel-Like Protein 1 (Mtmc1) Is Translated from a Splice Variant mTmc1ex1 but Not from the Other Variant mTmc1ex2.

Mechanical stimuli caused by sound waves are detected by hair cells in the cochlea through the opening of mechanoelectrical transduction (MET) channels. Transmembrane channel-like protein 1 (TMC1) has been revealed to be the pore-forming component of the MET channel. The two splice variants for mouse Tmc1 (mTmc1ex1 and mTmc1ex2) were reported to be expressed in the cochlea of infant mice, though only the sequence of mTmc1ex2 had been deposited in GenBank. However, due to the presence of an upstream open reading frame (uORF) and the absence of a typical Kozak sequence in mTmc1ex2, we questioned whether mTMC1 was translated from mTmc1ex2. Therefore, in this study, we evaluated which splice variant was protein-coding mRNA. Firstly, the results of RT-PCR and cDNA cloning of mTmc1 using mRNA isolated from the cochlea of five-week-old mice suggested that more Tmc1ex1 were expressed than mTmc1ex2. Secondly, mTMC1 was translated from mTmc1ex1 but not from mTmc1ex2 in a heterologous expression system. Finally, analyses using site-directed mutagenesis revealed that the uORF and the weak Kozak sequence in mTmc1ex2 prevented the translation of mTMC1 from mTmc1ex2. These results suggest that mTmc1ex1 plays a main role in the expression of mTMC1 in the mouse cochlea, and therefore, mTmc1ex1 should be the mRNA for mTMC1 hereafter.

Fibroblast growth factor 2 modulates extracellular purine metabolism by upregulating ecto-5'-nucleotidase and adenosine deaminase in cultured rat spinal cord astrocytes.

Purinergic signaling via ATP and adenosine produced by astrocytes is one pathway underlying neuron-glia interactions in the central nervous system (CNS). In production of purines, extracellular metabolism of released purines via ecto-enzymes is important. The expression and activities of these enzymes are altered under pathological conditions. Production of fibroblast growth factor 2 (FGF2) is increased under pathological conditions, and this has various effects on astrocytes. Here, we investigated the effects of FGF2 on purine metabolism in cultured rat spinal cord astrocytes. Astrocytes rapidly metabolized purines added to the extracellular solution. FGF2 increased extracellular metabolism of AMP to adenosine and of adenosine to inosine by upregulating ecto-5'-nucleotidase and adenosine deaminase (ADA), respectively. ADA activity and protein were detected both in the cytosol and external solution of astrocytes, and their levels were markedly increased by FGF2. FGF2 also increased metabolism of endogenously released ATP, resulting in a transient increase in adenosine and substantial accumulation of extracellular inosine. Moreover, FGF2 increased ATP release by upregulating the activity of gap junction hemichannels. These data show that FGF2 regulates purine production in astrocytes and suggest that extracellular ADA released by astrocytes plays an important role in extracellular purine metabolism in the CNS.

The α2A -adrenoceptor subtype plays a key role in the analgesic and sedative effects of xylazine.

Xylazine, the classical α2 -adrenoceptor (α2 -AR) agonist, is still used as an analgesic and sedative in veterinary medicine, despite its low potency and affinity for α2 -ARs. Previous pharmacological studies suggested that the α2A -AR subtype plays a role in mediating the clinical effects of xylazine; however, these studies were hampered by the poor subtype-selectivity of the antagonists used and a lack of knowledge of their bioavailability in vivo. Here, we attempted to elucidate the role of the α2A -AR subtype in mediating the clinical effects of xylazine by comparing the analgesic and sedative effects of this drug in wild-type mice with those in α2A -AR functional knockout mice using the hot-plate and open field tests, respectively. Hippocampal noradrenaline turnover in both mice was also measured to evaluate the contribution of α2A -AR subtype to the inhibitory effect of xylazine on presynaptic noradrenaline release. In wild-type mice, xylazine (10 or 30 mg/kg) increased the hot-plate latency. Furthermore, xylazine (3 or 10 mg/kg) inhibited the open field locomotor activity and decreased hippocampal noradrenaline turnover. By contrast, all of these effects were abolished in α2A -AR functional knockout mice. These results indicate that the α2A -AR subtype is mainly responsible for the clinical effects of xylazine.

TRPM4 and TRPM5 Channels Share Crucial Amino Acid Residues for Ca2+ Sensitivity but Not Significance of PI(4,5)P2.

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.

Negatively charged amino acids near and in transient receptor potential (TRP) domain of TRPM4 channel are one determinant of its Ca2+ sensitivity.

Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+).

Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes.

Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells.

Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons.

Hydrogen sulfide (H2 S) is a gaseous neuromodulator produced from L-cysteine. H2 S is generated by three distinct enzymatic pathways mediated by cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H2 S production in PC12 cells (rat pheochromocytoma-derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H2 S were produced from L-cysteine in the presence of α-ketoglutarate, together with dithiothreitol. The production of H2 S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L-aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H2 S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H2 S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H2 S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H2 S generation. In the peripheral nervous system, hydrogen sulfide (H2 S) has been implicated in neurogenic pain or hyperalgesia. This study provides evidence that H2 S is synthesized in peripheral neurons through two mitochondrial enzymes, cysteine aminotransferase (CAT) and mercaptopyruvate sulfurtransferase (MPST). We propose that mitochondrial metabolism plays key roles in the physiology and pathophysiology of the peripheral nervous system via regulation of neuronal H2 S production.

Endovascular treatment of acute atherothrombotic internal carotid artery occlusion associated with persistent primitive hypoglossal artery.

Persistent primitive hypoglossal artery is a relatively rare anatomical variation and a type of persistent carotid-basilar anastomosis. Acute internal carotid artery occlusion associated with persistent primitive hypoglossal artery is rare, and atherothrombotic occlusion is extremely rare. We present a case of acute atherothrombotic internal carotid artery occlusion associated with persistent primitive hypoglossal artery that was successfully treated by endovascular treatment. A 70-year-old male with a history of left internal carotid artery stenosis was transferred to our hospital by ambulance because of abnormal behaviors and aphasia. He was diagnosed with cerebral infarction and left internal carotid artery occlusion. Left carotid angiography revealed the persistent primitive hypoglossal artery arising from the cervical internal carotid artery and complete internal carotid artery occlusion distal to the origin of the persistent primitive hypoglossal artery. Therefore, we performed endovascular treatment. Mechanical thrombectomy was performed under minimal flow arrest with consideration of brain ischemia causing coma. After additional balloon angioplasty, recanalization was achieved, and the patient's symptoms improved. During the 1.5-year follow-up period, no recurrence or restenosis was observed. This report provides evidence that atherosclerotic internal carotid artery stenosis associated with persistent primitive hypoglossal artery can occur even distal to the origin of the persistent primitive hypoglossal artery and that the lesion may become acutely occluded, leading to acute stroke. Endovascular treatment considering brain ischemia was effective in this case.

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.

The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells.

Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic N-terminal (Nt) region of heterologously-expressed mouse TMC1 (mTMC1) was localized in nuclei of a small population of the transfected HEK293 cells. This raised the possibility that the Nt region of heterologously-expressed mTMC1 was cleaved and transported into the nucleus. To confirm the cleavage, we performed western blot analyses. The results revealed that at least a fragment of the Nt region was produced from heterologously-expressed mTMC1. Site-directed mutagenesis experiments identified amino acid residues which were required to produce the fragment. The accumulation of the heterologously-expressed Nt fragment into the nuclei depended on nuclear localization signals within the Nt region. Furthermore, a structural comparison showed a similarity between the Nt region of mTMC1 and basic region leucine zipper (bZIP) transcription factors. However, transcriptome analyses using a next-generation sequencer showed that the heterologously-expression of the Nt fragment of mTMC1 hardly altered expression levels of genes. Although it is still unknown what is the precise mechanism and the physiological significance of this cleavage, these results showed that the cytosolic Nt region of heterologously-expressed mTMC1 could be cleaved in HEK293 cells. Therefore, it should be taken into account that the cleavage of Nt region might influence the functional analysis of TMC1 by the heterologous-expression system using HEK293 cells.

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels.

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.

IRBIT reduces the apparent affinity for intracellular Mg²âº in inhibition of the electrogenic Na⁺-HCO3⁻ cotransporter NBCe1-B.

The electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1-B can be regulated by intracellular Mg(2+) (Mg(2+)(i)). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg(2+)(i), and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg(2+)(i) inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg(2+)(i) sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg(2+)(i) in inhibition of NBCe1-B activity in mammalian cells.

Posterior reversible encephalopathy syndrome mimicking subacute ischemic stroke: a case report.

Posterior reversible encephalopathy syndrome, an acute onset neurological syndrome, is among the conditions that must be differentiated from stroke. Herein, we report a rare case of posterior reversible encephalopathy syndrome mimicking subacute ischemic stroke. A 68-year-old man was transferred by ambulance to our hospital because of visual disturbance. He showed left homonymous hemianopsia. Magnetic resonance imaging (diffusion-weighted imaging and fluid-attenuated inversion recovery imaging) revealed high signal intensity in the right occipital lobe. We suspected subacute cerebral infarction. After admission, he developed cortical blindness and increased blood pressure. Fluid-attenuated inversion recovery imaging revealed high signal intensity and elevated apparent diffusion coefficient values in the bilateral occipital lobes. We diagnosed the patient with posterior reversible encephalopathy syndrome. Antihypertensive treatment improved his clinical symptoms. Careful imaging assessment, including of changes over time, is important for diagnosing posterior reversible encephalopathy syndrome.

Properties of the TRPML3 channel pore and its stable expansion by the Varitint-Waddler-causing mutation.

TRPML3 is a H(+)-regulated Ca(2+) channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca(2+) to change its conductance and permeability, which appears to be mediated by trapping Ca(2+) within the pore. The pore properties can be restored by strong depolarization or by conducting Na(+) through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca(2+). This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.

Hydrogen sulfide induces Ca2+ release from the endoplasmic reticulum and suppresses ATP-induced Ca2+ signaling in rat spinal cord astrocytes.

Hydrogen sulfide (H2S) has a variety of physiological functions. H2S reportedly increases intracellular Ca2+ concentration ([Ca2+]i) in astrocytes. However, the precise mechanism and functional role of this increase are not known. Here, we examined the effects of H2S on [Ca2+]i in astrocytes from the rat spinal cord and whether H2S affects ATP-induced Ca2+ signaling, which is known to be involved in synaptic function. Na2S (150 µM), an H2S donor, produced a nontoxic increase in [Ca2+]i. The [Ca2+]i increase by Na2S was inhibited by Ca2+ depletion in the endoplasmic reticulum (ER) but not by removal of extracellular Ca2+, indicating that H2S releases Ca2+ from the ER. On the other hand, Na2S inhibited ATP-induced [Ca2+]i increase when Na2S clearly increased [Ca2+]i in the astrocytes, which was not suppressed by a reducing agent. In addition, Na2S had no effect on intracellular cyclic AMP (cAMP) level. These results indicate that oxidative post-translational modification of proteins and cAMP are not involved in the inhibitory effect of H2S on ATP-induced Ca2+ signaling. We conclude that H2S indirectly inhibits ATP-induced Ca2+ signaling by decreasing Ca2+ content in the ER in astrocytes. In this way, H2S may influence intercellular communication between astrocytes and neurons, thereby contributing to neuronal signaling in the nervous system.

Functional characterization of a ClC-2-like Cl(-) conductance in surface epithelial cells of rat rectal colon.

ClC-2, a member of the voltage-gated Cl(-) channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na(+) conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl(-) current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl(-) current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn(2+) sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn(2+) (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl(-) channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na(+) transport in this surface epithelium under basal physiological conditions.