Sigma-1 receptor is involved in modification of ER-mitochondria proximity and Ca2+ homeostasis in cardiomyocytes.

The Sigma-1 receptor (Sigmar1) is downregulated in heart failure model mice with mitochondrial dysfunction. However, the mechanism in detail has not been investigated. In this study, we investigated the role of Sigmar1 in ER-mitochondria proximity using Sigmar1-knockdown or -overexpressed neonatal rat ventricular myocytes (NRVMs). The endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy was aggravated with the dysregulation of mitochondrial function and ER-mitochondrial junctional formation in Sigmar1-knockdown NRVMs, whereas improved in Sigmar1 overexpressed NRVMs. Our data suggests that the reduction of the cardiac Sigmar1 results in decrease mitochondrial Ca2+ influx and promotes mitochondrial fission, followed by reduced ER-mitochondria proximity, exacerbating ET-1-induced cardiomyocyte injury.

Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties.

There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.

Vasopressin Neurons Respond to Hyperosmotic Stimulation with Regulatory Volume Increase and Secretory Volume Decrease by Activating Ion Transporters and Ca2+ Channels.

BACKGROUND/AIMS: Arginine vasopressin (AVP) neurons play an important role for sensing a change in the plasma osmolarity and thereby responding with regulated AVP secretion in order to maintain the body fluid homeostasis. The osmo-sensing processes in magnocellular neurosecretory cells (MNCs) including AVP and oxytocin (OXT) neurons of the hypothalamus were reported to be coupled to sustained osmotic shrinkage or swelling without exhibiting discernible cell volume regulation. Since increasing evidence has shown some important differences in properties between AVP and OXT neurons, osmotic volume responses are to be reexamined with distinguishing these cell types from each other. We previously reported that AVP neurons identified by transgenic expression of enhanced green fluorescence protein (eGFP) possess the ability of regulatory volume decrease (RVD) after hypoosmotic cell swelling. Thus, in the present study, we examined the ability of regulatory volume increase (RVI) after hyperosmotic cell shrinkage in AVP neurons. METHODS: Here, we used eGFP-identified AVP neurons acutely dissociated from AVP-eGFP transgenic rats. We performed single-cell size measurements, cytosolic RT-PCR analysis, AVP secretion measurements, and patch-clamp studies. RESULTS: The AVP neurons were found to respond to a hyperosmotic challenge with physiological cell shrinkage caused by massive secretion of AVP, called a secretory volume decrease (SVD), superimposed onto physical osmotic cell shrinkage, and also to exhibit the ability of RVI coping with osmotic and secretory cell shrinkage. Furthermore, our pharmacological and molecular examinations indicated that AVP secretion and its associated SVD event are triggered by activation of T-type Ca2+ channels, and the RVI event is attained by parallel operation of Na+/H+ exchanger and Cl-/HCO3- anion exchanger. CONCLUSION: Thus, it is concluded that AVP neurons respond to hyperosmotic stimulation with the regulatory volume increase and the secretory volume increase by activating ion transporters and Ca2+ channels, respectively.

Elucidation of the Mechanisms for the Underlying Depolarization and Reversibility by Photoactive Molecule.

BACKGROUND/AIMS: Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. METHODS: Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. RESULTS: Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. CONCLUSION: Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation.

Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 µM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.

Short TRPM2 prevents the targeting of full-length TRPM2 to the surface transmembrane by hijacking to ER associated degradation.

Membrane proteins are targeted to the surface transmembrane after folding and assembling in the endoplasmic reticulum (ER). Misfolded- and unassembled-proteins are degraded by proteasomes following ubiquitination, termed ER-associated degradation (ERAD). Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive channel. One of the TRPM2 splicing variants, short TRPM2 (TRPM2-S) having only the N-terminus and first two transmembrane domains, was reported to prevent full-length TRPM2 (TRPM2-L) activation. Although TRPM2-S interacts with TRPM2-L, the inhibitory mechanisms of TRPM2-S are unclear. We found that TRPM2-S prevents transmembrane expression of TRPM2-L by targeting ERAD. TRPM2-S expression was lower than that of TRPM2-L, and was increased by an ERAD inhibitor. TRPM2-S was not expressed at the transmembrane. This suggests that TRPM2-S is a substrate for ERAD. Upon the simultaneous expression of TRPM2-S, the transmembrane expression of TRPM2-L was attenuated and the poly-ubiquitination of TRPM2-L was facilitated. Our study may clarify why TRPM2-S inhibits oxidative stress-induced TRPM2-L activation.

Roles of volume-regulatory anion channels, VSOR and Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death, stroke and myocardial infarction.

Two types of anion channels are directly activated by osmotic swelling and are involved in the regulatory volume decrease (RVD) in most types of mammalian cells, and they include the volume-sensitive outwardly rectifying anion channel (VSOR), also called the volume-regulated anion channel (VRAC), and the large-conductance maxi-anion channel (Maxi-Cl). In cardiomyocytes, a splice variant of cystic fibrosis transmembrane conductance regulator anion channel (cardiac CFTR) participates in the RVD mechanism under ß-adrenergic stimulation. VSOR and Maxi-Cl are also involved in facilitation of the RVD process by releasing extracellular autocrine/paracrine signals, glutamate and ATP. Apoptotic cell death starts with cell shrinkage, called the apoptotic volume decrease (AVD), which is also caused by activation of VSOR. Since VSOR is implicated not only in the AVD induction but also in the uptake of an anti-cancer drug, cisplatin, downregulation of VSOR activity is causatively involved in acquisition of cisplatin resistance in cancer cells. Necrotic cell death exhibits persistent cell swelling, called the necrotic volume increase (NVI), which is coupled to RVD dysfunction due to impaired VSOR function. Acidotoxic and lactacidosis-induced necrotic cell death is induced both by glutamate release mediated by astroglial VSOR and Maxi-Cl and by exaggerated Cl- influx mediated by neuronal VSOR under prolonged depolarization caused by activation of ionotropic glutamate receptor (iGluR) cation channels. Both VSOR and Maxi-Cl are elaborately involved, in a manner as double-edged swords, in ischemia- and ischemia-reperfusion-induced apoptotic or necrotic cell death in cerebral and myocardial cells by mediating not only Cl- transport but also release of glutamate and/or ATP. Cardiac CFTR exerts a protective action against ischemia(-reperfusion)-induced cardiac injury, called myocardial infarction (MI), which is largely necrotic. Cardiac Maxi-Cl activity may participate in protection against ischemia(-reperfusion) injury by mediating ATP release.

Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins.

Distinct pharmacological and molecular properties of the acid-sensitive outwardly rectifying (ASOR) anion channel from those of the volume-sensitive outwardly rectifying (VSOR) anion channel.

Expressed by many cell types, acid-sensitive outwardly rectifying (ASOR) anion channels are known to be activated by extracellular acidification and involved in acidotoxic necrotic cell death. In contrast, ubiquitously expressed volume-sensitive outwardly rectifying (VSOR) anion channels are activated by osmotic cell swelling and involved in cell volume regulation and apoptotic cell death. Distinct inhibitors to distinguish ASOR from VSOR anion channels have not been identified. Although leucine-rich repeats containing 8A (LRRC8A) was recently found to be an essential component of VSOR anion channels, the possibility of an LRRC8 family member serving as a component of ASOR anion channels has not been examined. In this study, we explored the effects of 12 known VSOR channel inhibitors and small interfering RNA (siRNA)-mediated knockdown of LRRC8 family members on ASOR and VSOR currents in HeLa cells. Among these inhibitors, eight putative VSOR blockers, including 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), were totally ineffective at blocking ASOR channel activity, whereas suramin, R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] acetic acid (DIOA), arachidonic acid, and niflumic acid were found to be effective ASOR anion channel antagonists. In addition, gene-silencing studies showed that no LRRC8 family members are essentially involved in ASOR anion channel activity, whereas LRRC8A is involved in VSOR anion channel activity in HeLa cells.

Compromised maturation of GABAergic inhibition underlies abnormal network activity in the hippocampus of epileptic Ca2+ channel mutant mice, tottering.

Cholinergically induced network activity is a useful analogue of theta rhythms involved in memory processing or epileptiform activity in the hippocampus, providing a powerful tool to elucidate the mechanisms of synchrony in neuronal networks. In absence epilepsy, although its association with cognitive impairments has been reported, the mechanisms underlying hippocampal synchrony remain poorly investigated. Here we simultaneously recorded electrical activities from 64 sites in hippocampal slices of CaV2.1 Ca(2+) channel mutant tottering (tg) mice, a well-established mouse model of spontaneous absence epilepsy, to analyze the spatiotemporal pattern of cholinergically induced hippocampal network activity. The cholinergic agonist carbachol induced oscillatory discharges originating from the CA3 region. In tg/tg mice, this hippocampal network activity was characterized by enhanced occupancy of discharges of relatively high frequency (6-10 Hz) compared to the wild type. Pharmacological analyses of slices, patch clamp electrophysiological characterization of isolated neurons, and altered patterns of hippocampal GABAA receptor subunit and Cl(-) transporter messenger RNA (mRNA) transcript levels revealed that this abnormality is attributable to a developmental retardation of GABAergic inhibition caused by immature intracellular Cl(-) regulation. These results suggest that the inherited CaV2.1 Ca(2+) channel mutation leads to developmental abnormalities in Cl(-) transporter expression and GABAA receptor compositions in hippocampal neurons and that compromised maturation of GABAergic inhibition contributes to the abnormal synchrony in the hippocampus of tg absence epileptic mice.

Thermosensitive Ion Channel Activation in Single Neuronal Cells by Using Surface-Engineered Plasmonic Nanoparticles.

Controlling cell functions using external photoresponsive nanomaterials has enormous potential for the development of cell-engineering technologies and intractable disease therapies, but the former currently requires genetic modification of the target cells. We present a method using plasma-membrane-targeted gold nanorods (pm-AuNRs) prepared with a cationic protein/lipid complex to activate a thermosensitive cation channel, TRPV1, in intact neuronal cells. Highly localized photothermal heat generation mediated by the pm-AuNRs induced Ca(2+) influx solely by TRPV1 activation. In contrast, the use of previously reported cationic AuNRs that are coated with a conventional synthetic polymer also led to photoinduced Ca(2+) influx, but this influx resulted from membrane damage. Our method provides an optogenetic platform without the need for prior genetic engineering of the target cells and might be useful for novel TRPV1-targeted phototherapeutic approaches.

Transnitrosylation directs TRPA1 selectivity in N-nitrosamine activators.

S-Nitrosylation, the addition of a nitrosyl group to cysteine thiols, regulates various protein functions to mediate nitric oxide (NO) bioactivity. Recent studies have demonstrated that selectivity in protein S-nitrosylation signaling pathways is conferred through transnitrosylation, a transfer of the NO group, between proteins via interaction. We previously demonstrated that sensitivity to activation by synthetic NO-releasing agents via S-nitrosylation is a common feature of members of the transient receptor potential (TRP) family of Ca(2+)-permeable cation channels. However, strategies to confer subtype selectivity to nitrosylating agents targeted to TRP channels are yet to be developed. Here, we show selective activation of TRPA1 channels by novel NO donors derived from the ABBH (7-azabenzobicyclo[2.2.1]heptane) N-nitrosamines, which exhibit transnitrosylation reactivity to thiols without releasing NO. The NNO-ABBH1 (N-nitroso-2-exo,3-exo-ditrifluoromethyl-7-azabenzobicyclo[2.2.1]heptane) elicits S-nitrosylation of TRPA1 proteins, and dose-dependently induces robust Ca(2+) influx via both recombinant and native TRPA1 channels, but not via other NO-activated TRP channels. TRPA1 activation by NNO-ABBH1 is suppressed by specific cysteine mutations but not by NO scavenging, suggesting that cysteine transnitrosylation underlies the activation of TRPA1 by NNO-ABBH1. This is supported by the correlation of N-NO bond reactivity and TRPA1-activating potency in a congeneric series of ABBH N-nitrosamines. Interestingly, nonelectrophilic derivatives of ABBH also activate TRPA1 selectively, but less potently, compared with NNO-ABBH1. Thus, ABBH N-nitrosamines confer subtype selectivity on S-nitrosylation in TRP channels through synergetic effects of two chemical processes: cysteine transnitrosylation and molecular recognition of the nonelectrophilic moiety.

Transient receptor potential melastatin 2 protects mice against polymicrobial sepsis by enhancing bacterial clearance.

BACKGROUND: Recent studies suggest that the transient receptor potential melastatin 2 (TRPM2) channel plays an important role in inflammation and immune response. However, the role and mechanism of TRPM2 in polymicrobial sepsis remain unclear. METHODS: The authors explored the effects of genetic disruption of TRPM2 on mortality (n = 15), bacterial clearance (n = 6), organ injury, and systemic inflammation during cecal ligation and puncture-induced sepsis. Electrophysiology, immunoblot, bacterial clearance experiment, and quantitative real-time polymerase chain reaction were used to explore the role and mechanism of TRPM2 in sepsis. RESULTS: After cecal ligation and puncture, Trpm2-knockout mice had increased mortality compared with wild-type mice (73.3 vs. 40%, P = 0.0289). The increased mortality was associated with increased bacterial burden, organ injury, and systemic inflammation. TRPM2-mediated Ca influx plays an important role in lipopolysaccharide or cecal ligation and puncture-induced heme oxygenase-1 (HO-1) expression in macrophage. HO-1 up-regulation decreased bacterial burden both in wild-type bone marrow-derived macrophages and in cecal ligation and puncture-induced septic wild-type mice. Disruption of TRPM2 decreased HO-1 expression and increased bacterial burden in bone marrow-derived macrophages. Pretreatment of Trpm2-knockout bone marrow-derived macrophages with HO-1 inducer markedly increased HO-1 expression and decreased bacterial burden. Pretreatment of Trpm2-knockout mice with HO-1 inducer reversed the susceptibility of Trpm2-knockout mice to sepsis by enhancing the bacterial clearance. In addition, septic patients with lower monocytic TRPM2 and HO-1 messenger RNA levels had a worse outcome compared with septic patients with normal monocytic TRPM2 and HO-1 messenger RNA levels. TRPM2 levels correlated with HO-1 levels in septic patients (r = 0.675, P = 0.001). CONCLUSION: The study data demonstrate a protective role of TRPM2 in controlling bacterial clearance during polymicrobial sepsis possibly by regulating HO-1 expression.

TRP channels as sensors of oxygen availability.

An ability to adapt to changes in oxygen availability is essential for survival in both prokaryotic and eukaryotic organisms. Recently, cation channels encoded by the transient receptor potential (trp) gene superfamily have been recognized as multimodal sensors of a wide variety of factors inside the cells and in the extracellular environment and also as transducers of electrical and chemical signals mediated by ions such as Ca(2+). The functional features of TRP channels enable the body to react and adapt to different forms of environmental changes, including oxygen levels. A subclass of TRP channels regulates various cellular processes in response to fluctuations in oxygen. In this article, we describe the physiological and pathological significance of the oxygen-sensitive TRP channels, which are heterogeneous in the cellular responses to acute changes in oxygen, by contrasting their oxygen monitoring function with that of other ion channels, transporters, and enzymes. We also discuss the physiological relevance of oxygen-sensitive TRP channels as a novel class of target proteins for pharmaceutical therapeutics.

Acid-sensitive outwardly rectifying (ASOR) anion channels in human epithelial cells are highly sensitive to temperature and independent of ClC-3.

A novel type of anion channel activated by extracellular acidification, called acid-sensitive outwardly rectifying (ASOR) anion channel, was shown to be involved in acidotoxic necrotic death in human epithelial cells. However, its biophysical property and molecular identity have remained elusive. In human epithelial HeLa cells, here, whole-cell currents of ASOR anion channel were found to be augmented by warm temperature, with a threshold temperature of 32 °C. Temperature sensitivity of the conductance was found to be high (with Q 10 of 8.8) in the range of body temperature, suggesting a possible involvement of a non-diffusion-limited process such as a transporter-mediated conduction. In this regard, it is interesting that a Cl(-)/H(+) antiporter ClC-3 has recently been proposed as a candidate for the ASOR channel. However, siRNA-mediated knockdown of hClC-3 failed to suppress ASOR currents in HeLa cells. Also, endogenous ASOR currents in HEK293T cells were not affected by overexpression of human or mouse ClC-3. Furthermore, functional expression of the ASOR channel was virtually absent in the cisplatin-resistant human cancer KCP-4 cell line despite the fact that molecular expression of ClC-3 was indistinguishable between KCP-4 cells and parental cisplatin-sensitive KB-3-1 cells which endogenously exhibit high activity of ASOR anion channels. These results indicate that the ASOR anion channel is highly sensitive to temperature and independent of ClC-3.

TRPA1 underlies a sensing mechanism for O2.

Oxygen (O(2)) is a prerequisite for cellular respiration in aerobic organisms but also elicits toxicity. To understand how animals cope with the ambivalent physiological nature of O(2), it is critical to elucidate the molecular mechanisms responsible for O(2) sensing. Here our systematic evaluation of transient receptor potential (TRP) cation channels using reactive disulfides with different redox potentials reveals the capability of TRPA1 to sense O(2). O(2) sensing is based upon disparate processes: whereas prolyl hydroxylases (PHDs) exert O(2)-dependent inhibition on TRPA1 activity in normoxia, direct O(2) action overrides the inhibition via the prominent sensitivity of TRPA1 to cysteine-mediated oxidation in hyperoxia. Unexpectedly, TRPA1 is activated through relief from the same PHD-mediated inhibition in hypoxia. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and hypoxia-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O(2)-sensing mechanism mediated by TRPA1.

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade.

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.

Mutations in EFHC1 cause juvenile myoclonic epilepsy.

Juvenile myoclonic epilepsy (JME) is the most frequent cause of hereditary grand mal seizures. We previously mapped and narrowed a region associated with JME on chromosome 6p12-p11 (EJM1). Here, we describe a new gene in this region, EFHC1, which encodes a protein with an EF-hand motif. Mutation analyses identified five missense mutations in EFHC1 that cosegregated with epilepsy or EEG polyspike wave in affected members of six unrelated families with JME and did not occur in 382 control individuals. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly lowered by the mutations. Apoptosis was specifically suppressed by SNX-482, an antagonist of R-type voltage-dependent Ca(2+) channel (Ca(v)2.3). EFHC1 and Ca(v)2.3 immunomaterials overlapped in mouse brain, and EFHC1 coimmunoprecipitated with the Ca(v)2.3 C terminus. In patch-clamp analysis, EFHC1 specifically increased R-type Ca(2+) currents that were reversed by the mutations associated with JME.

Cell death induction and protection by activation of ubiquitously expressed anion/cation channels. Part 3: the roles and properties of TRPM2 and TRPM7.

Cell volume regulation (CVR) is a prerequisite for animal cells to survive and fulfill their functions. CVR dysfunction is essentially involved in the induction of cell death. In fact, sustained normotonic cell swelling and shrinkage are associated with necrosis and apoptosis, and thus called the necrotic volume increase (NVI) and the apoptotic volume decrease (AVD), respectively. Since a number of ubiquitously expressed ion channels are involved in the CVR processes, these volume-regulatory ion channels are also implicated in the NVI and AVD events. In Part 1 and Part 2 of this series of review articles, we described the roles of swelling-activated anion channels called VSOR or VRAC and acid-activated anion channels called ASOR or PAC in CVR and cell death processes. Here, Part 3 focuses on therein roles of Ca2+-permeable non-selective TRPM2 and TRPM7 cation channels activated by stress. First, we summarize their phenotypic properties and molecular structure. Second, we describe their roles in CVR. Since cell death induction is tightly coupled to dysfunction of CVR, third, we focus on their participation in the induction of or protection against cell death under oxidative, acidotoxic, excitotoxic, and ischemic conditions. In this regard, we pay attention to the sensitivity of TRPM2 and TRPM7 to a variety of stress as well as to their capability to physicall and functionally interact with other volume-related channels and membrane enzymes. Also, we summarize a large number of reports hitherto published in which TRPM2 and TRPM7 channels are shown to be involved in cell death associated with a variety of diseases or disorders, in some cases as double-edged swords. Lastly, we attempt to describe how TRPM2 and TRPM7 are organized in the ionic mechanisms leading to cell death induction and protection.

Cardioprotective effects of Moku-boi-to and its impact on AngII-induced cardiomyocyte hypertrophy.

Cardiomyocyte hypertrophy, induced by elevated levels of angiotensin II (AngII), plays a crucial role in cardiovascular diseases. Current therapeutic approaches aim to regress cardiac hypertrophy but have limited efficacy. Widely used Japanese Kampo medicines are highly safe and potential therapeutic agents. This study aims to explore the impact and mechanisms by which Moku-boi-to (MBT), a Japanese Kampo medicine, exerts its potential cardioprotective benefits against AngII-induced cardiomyocyte hypertrophy, bridging the knowledge gap and contributing to the development of novel therapeutic strategies. By evaluating the effects of six Japanese Kampo medicines with known cardiovascular efficiency on AngII-induced cardiomyocyte hypertrophy and cell death, we identified MBT as a promising candidate. MBT exhibited preventive effects against AngII-induced cardiomyocyte hypertrophy, cell death and demonstrated improvements in intracellular Ca2+ signaling regulation, ROS production, and mitochondrial function. Unexpectedly, experiments combining MBT with the AT1 receptor antagonist losartan suggested that MBT may target the AT1 receptor. In an isoproterenol-induced heart failure mouse model, MBT treatment demonstrated significant effects on cardiac function and hypertrophy. These findings highlight the cardioprotective potential of MBT through AT1 receptor-mediated mechanisms, offering valuable insights into its efficacy in alleviating AngII-induced dysfunction in cardiomyocytes. The study suggests that MBT holds promise as a safe and effective prophylactic agent for cardiac hypertrophy, providing a deeper understanding of its mechanisms for cardioprotection against AngII-induced dysfunction.