Shin-Young Ryu

Single channel characterization of the mitochondrial ryanodine receptor in heart mitoplasts.

Heart mitochondria utilize multiple Ca2+ transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca2+ uptake pathway across the inner membrane to control “excitation and metabolism coupling.” In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ∼100, ∼225, ∼700, and ∼1,000 picosiemens (pS) in symmetrical 150 mm CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels.

Leptin promotes KATP channel trafficking by AMPK signaling in pancreatic β-cells

Leptin is a pivotal regulator of energy and glucose homeostasis, and defects in leptin signaling result in obesity and diabetes. The ATP-sensitive potassium (KATP) channels couple glucose metabolism to insulin secretion in pancreatic β-cells. In this study, we provide evidence that leptin modulates pancreatic β-cell functions by promoting KATP channel translocation to the plasma membrane via AMP-activated protein kinase (AMPK) signaling. KATP channels were localized mostly to intracellular compartments of pancreatic β-cells in the fed state and translocated to the plasma membrane in the fasted state. This process was defective in leptin-deficient ob/ob mice, but restored by leptin treatment. We discovered that the molecular mechanism of leptin-induced AMPK activation involves canonical transient receptor potential 4 and calcium/calmodulin-dependent protein kinase kinase β. AMPK activation was dependent on both leptin and glucose concentrations, so at optimal concentrations of leptin, AMPK was activated sufficiently to induce KATP channel trafficking and hyperpolarization of pancreatic β-cells in a physiological range of fasting glucose levels. There was a close correlation between phospho-AMPK levels and β-cell membrane potentials, suggesting that AMPK-dependent KATP channel trafficking is a key mechanism for regulating β-cell membrane potentials. Our results present a signaling pathway whereby leptin regulates glucose homeostasis by modulating β-cell excitability.

Distinctive characteristics and functions of multiple mitochondrial Ca2+ influx mechanisms

Intracellular Ca2+ is vital for cell physiology. Disruption of Ca2+ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca2+ dynamics, various Ca2+ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca2+ transport mechanisms in responding to physiological Ca2+ pulses in cytosol is to take up Ca2+ for regulating energy production and shaping the amplitude and duration of Ca2+ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca2+ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca2+ transport across the inner mitochondrial membrane. The mitochondrial Ca2+ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca2+ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca2+ influx. Since multiple Ca2+ influx mechanisms (e.g. L-, T-, and N-type Ca2+ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca2+ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca2+ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca2+/H+ exchanger), and MCU and its Ca2+ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca2+ influx pathways and their differences in kinetics, Ca2+ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.

Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

SIGNIFICANCE Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. RECENT ADVANCES Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. CRITICAL ISSUES Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. FUTURE DIRECTIONS Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca2+-activated K+ channels and inward rectifier K+ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.

Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca2+ current in cardiomyocytes

Oxidative stress remodels Ca(2+) signaling in cardiomyocytes, which promotes altered heart function in various heart diseases. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) was shown to be activated by oxidation, but whether and how CaMKII links oxidative stress to pathophysiological long-term changes in Ca(2+) signaling remain unknown. Here, we present evidence demonstrating the role of CaMKII in transient oxidative stress-induced long-term facilitation (LTF) of L-type Ca(2+) current (I(Ca,L)) in rat cardiomyocytes. A 5-min exposure of 1mM H(2)O(2) induced an increase in I(Ca,L), and this increase was sustained for ~1h. The CaMKII inhibitor KN-93 fully reversed H(2)O(2)-induced LTF of I(Ca,L), indicating that sustained CaMKII activity underlies this oxidative stress-induced memory. Simultaneous inhibition of oxidation and autophosphorylation of CaMKII prevented the maintenance of LTF, suggesting that both mechanisms contribute to sustained CaMKII activity. We further found that sarcoplasmic reticulum Ca(2+) release and mitochondrial ROS generation have critical roles in sustaining CaMKII activity via autophosphorylation- and oxidation-dependent mechanisms. Finally, we show that long-term remodeling of the cardiac action potential is induced by H(2)O(2) via CaMKII. In conclusion, CaMKII and mitochondria confer oxidative stress-induced pathological cellular memory that leads to cardiac arrhythmia.

Acredinones A and B, Voltage-Dependent Potassium Channel Inhibitors from the Sponge-Derived Fungus Acremonium sp. F9A015

Two new benzophenones, acredinones A (1) and B (2), were isolated from a marine-sponge-associated Acremonium sp. fungus. Their chemical structures were elucidated on the interpretation of spectroscopic data. The structure of 1 was confirmed by palladium-catalyzed hydrogenation, followed by spectroscopic data analysis. Acredinones A (1) and B (2) inhibited the outward K(+) currents of the insulin secreting cell line INS-1 with IC50 values of 0.59 and 1.0 μM, respectively.

Ca 2+ clearance by plasmalemmal NCLX, Li + -permeable Na + /Ca 2+ exchanger, is required for the sustained exocytosis in rat insulinoma INS-1 cells

Na+/Ca2+ exchangers are key players for Ca2+ clearance in pancreatic β-cells, but their molecular determinants and roles in insulin secretion are not fully understood. In the present study, we newly discovered that the Li+-permeable Na+/Ca2+ exchangers (NCLX), which were known as mitochondrial Na+/Ca2+ exchangers, contributed to the Na+-dependent Ca2+ movement across the plasma membrane in rat INS-1 insulinoma cells. Na+/Ca2+ exchange activity by NCLX was comparable to that by the Na+/Ca2+ exchanger, NCX. We also confirmed the presence of NCLX proteins on the plasma membrane using immunocytochemistry and cell surface biotinylation experiments. We further investigated the role of NCLX on exocytosis function by measuring the capacitance increase in response to repetitive depolarization. Small interfering (si)RNA-mediated downregulation of NCLX did not affect the initial exocytosis, but significantly suppressed sustained exocytosis and recovery of exocytosis. XIP (NCX inhibitory peptide) or Na+ replacement for inhibiting Na+-dependent Ca2+ clearance also selectively suppressed sustained exocytosis. Consistent with the idea that sustained exocytosis requires ATP-dependent vesicle recruitment, mitochondrial function, assessed by mitochondrial membrane potential (ΔΨ), was impaired by siNCLX or XIP. However, depolarization-induced exocytosis was hardly affected by changes in intracellular Na+ concentration, suggesting a negligible contribution of mitochondrial Na+/Ca2+ exchanger. Taken together, our data indicate that Na+/Ca2+ exchanger-mediated Ca2+ clearance mediated by NCLX and NCX is crucial for optimizing mitochondrial function, which in turn contributes to vesicle recruitment for sustained exocytosis in pancreatic β-cells.