Miyuki Kuno

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (Iratio) were determined. The use of Iratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q10s for the proton currents were evaluated from the Iratios. Q10 exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q10. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q10 was decomposed into Q10 of the channel and of the access resistances. Finally, the Q10 for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.

Regulatory mechanisms and physiological relevance of a voltage-gated H+ channel in murine osteoclasts: phorbol myristate acetate induces cell acidosis and the channel activation.

The voltage‐gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis.

Synergetic activation of outwardly rectifying Cl− currents by hypotonic stress and external Ca2+ in murine osteoclasts

1 An outwardly rectifying Cl− (ORCl) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). 2 The hypotonically activated ORCl current was inhibited by intracellular dialysis with an ATP‐free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5′‐O‐(3‐thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6–8.0. 3 Intracellular application of cytochalasin D occasionally activated the ORCl current without hypotonic stress, but inhibited activation of the ORCl current by hypotonic stimulation. The hypotonically activated ORCl current was unaffected by a non‐actin‐depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. 4 Removal of extracellular Ca2+ inhibited activation of the ORCl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated ORCl current was partially decreased by intracellular dialysis with 20 mm EGTA. 5 With 10 mm Ca2+ in the extracellular medium, the ORCl current was activated in response to more minor decreases in osmolarity than with 1 mm Ca2+. The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca2+ level (pCa 6.5). 6 These results suggest that hypotonic stimulation and a rise in the extracellular Ca2+ level synergistically activate the ORCl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).

Coupled K+–Water Flux through the HERG Potassium Channel Measured by an Osmotic Pulse Method

The streaming potential (V stream) is a signature feature of ion channels in which permeating ions and water molecules move in a single file. V stream provides a quantitative measure of the ion and water flux (the water–ion coupling ratio), the knowledge of which is a prerequisite for elucidating the mechanisms of ion permeation. We have developed a method to measure V stream with the whole-cell patch-clamp configuration. A HEK293 cell stably expressing the HERG potassium channel was voltage clamped and exposed to hyperosmotic solutions for short periods of time (<1 s) by an ultrafast solution switching system (the osmotic pulse [quick jump-and-away] method). The reversal potentials were monitored by a series of voltage ramps before, during, and after the osmotic pulse. The shifts of the reversal potentials immediately after the osmotic jump gave V stream. In symmetrical K+ solutions (10 mM), the V streams measured at different osmolalities showed a linear relationship with a slope of −0.7 mV/ΔOsm, from which the water–ion coupling ratio (n, the ratio of the flux of water to the flux of cations; Levitt, D.G., S.R. Elias, and J.M. Hautman. 1978. Biochim. Biophys. Acta. 512:436–451) was calculated to be 1.4. In symmetrical 100 mM K+ solutions, the coupling ratio was decreased significantly (n = 0.9), indicating that the permeation process through states with increased ion occupancy became significant. We presented a diagrammatic representation linking the water–ion coupling ratio to the mode of ion permeation and suggested that the coupling ratio of one may represent the least hydrated ion flux in the single-file pore.

The effects of general anesthetics on P2X7 and P2Y receptors in a rat microglial cell line.

BACKGROUND: Microglial cells play important roles in coordinating the inflammatory brain responses to hypoxia and trauma. Ionotropic P2X receptors and metabotropic P2Y receptors (P2YRs) expressed in microglia can be activated by extracellular adenosine triphosphate (ATP) derived from damaged cells or astrocytes, and participate in the signaling pathways evoked in brain insult. Although several inhaled and IV anesthetics produce neuroprotective effects through neuronal mechanisms, little is known about how general anesthetics modulate microglial responses in the pathological state. We examined the effects of various general anesthetics on purinergic responses in a rat microglial cell line. METHODS: Currents were consistently activated by applications of ATP via a U-tube system under the whole-cell configuration. ATP-induced nondesensitizing currents observed after several applications of ATP exhibited characteristics of P2X7 receptors. The P2YRs-mediated mobilization of intracellular Ca2+ was measured using a Ca2+-sensitive fluorescent dye (fura-2). RESULTS: Inhaled anesthetics (sevoflurane, isoflurane, and halothane) at doses three times as high as minimum alveolar concentrations had no effect on the P2X7Rs-mediated currents. IV anesthetics (ketamine, propofol, and thiopental) enhanced the P2X7Rs-mediated currents reversibly. The potencies for activation of P2X7Rs were not correlated with the octanol/buffer partition coefficients. Thiopental, at low concentrations, slightly inhibited the P2X7Rs-mediated currents, suggesting its dual actions on P2X7Rs. The P2YRs-mediated mobilization of intracellular Ca2+ was not affected by any of the general anesthetics tested. CONCLUSIONS: Our results suggest that IV anesthetics, particularly thiopental and propofol, may modulate microglial functions through P2X7Rs in pathological conditions.

Temporal fluctuations of voltage-gated proton currents in rat spinal microglia via pH-dependent and -independent mechanisms

Voltage-gated proton (H(+)) channels are unique mechanisms to extrude a massive amount of H(+), and are proposed to regulate intracellular pH of microglia during respiratory bursts. Temporal variations of the H(+) current were studied in rat spinal microglia cultivated on the glial cell layer using the voltage-ramp protocol. Repetitive applications of the large and long-lasting depolarization decreased the amplitudes of the H(+) current transiently and reversibly. This decrease was accompanied by a shift of the reversal potential to a more positive direction, indicating that a drop in the transmembrane pH gradient (delta pH) by the H(+) efflux through the channel reduced the current. The decline of the H(+) current during depolarizations was also observed in a rat microglial cell line (GMI-R1). An increase in the extracellular buffer suppressed the reduction of the current, suggesting that H(+) secreted into the extracellular space contributed to the drop in delta pH. On the other hand, the amplitudes of the H(+) current often fluctuated greatly at intervals of 5-20 min without changes in delta pH. These results suggest that the H(+) current of microglia is tuned via both delta pH-dependent and -independent mechanisms, which may regulate both microglial behavior and the pH environments of the surrounding neural tissue.

Mast cell degranulating (MCD) peptide and its optical isomer activate GTP binding protein in rat mast cells

The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx‐sensitive G‐protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment. but soon returned to the original level. D‐MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx‐sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G‐protein than the α‐helix conformation.

Sites and mechanisms of action of lidocaine upon the isolaled spinal cord of the frog

The sites of action of lidocaine on the responses evoked by stimulation of lateral column (LC) and dorsal root (DR) were studied in the isolated, intra-arterially perfused spinal cord of the bullfrog. When the ventral root volley produced by stimulation was abolished by lidocaine, the presynaptic focal potential was almost unchanged. Intracellular recordings from motoneurons clearly demonstrated a marked reduction in amplitude of the EPSPs before the block of conduction of presynaptic fibers and the block of invasion of the neuron soma by antidromic spike potential. At low concentrations of lidocaine, the EPSPs elicited by LC stimulation produced shortening in time to peak, slowing in the decay time, decrease in amplitude and smaller changes in the later EPSPs of a train than the earlier ones. From the observations, it was concluded that the low concentrations of lidocaine affected primarily synaptic transmission in the spinal cord. The possible mechanisms of action of lidocaine were discussed.

Phospholipase C-dependent Ca2+-sensing pathways leading to endocytosis and inhibition of the plasma membrane vacuolar H+-ATPase in osteoclasts.

In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance (C(m)) simultaneously. C(m) is a measure of cell surface. Extracellular Ca2+ (2.5-40 mM) decreased C(m) and the V-ATPase current simultaneously. The decreased C(m), together with the enhanced uptake of a lipophilic dye (FM1-43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A(1), a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C gamma2 subunit. Holding the cytosolic Ca2+ at either high (0.5-5 microM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.

Efferent discharges of sympathetic and parasympathetic nerve fibers during increased intracranial pressure in anesthetized cats in the absence and presence of pressor response

Efferent discharges of the cervical sympathetic cardiovascular and vagal type 1 fibers in response to increased intracranial pressure (ICP) were simultaneously recorded in cats anesthetized with pentobarbitone and ventilated artificially. Sympathetic outflow of renal nerve fibers was also recorded in some animals. The type 1 fibers were assumed to be cardiac vagal fibers, from the response behavior such a pulse-synchronicity to respiratory and heart rhythm, reflex activation from arterial baroreceptors and reciprocal relationship of the activity to sympathetic ones during slower fluctuations of hemodynamic changes, and which occur spontaneously during Mayer waves. The vagal type 1 discharges increased to various amplitudes with increase in ICP and in the absence and the presence of pressor response. Efferent outflow of the renal and cervical sympathetic fibers frequently decreased with a moderate increase in ICP. There was a slight decrease or no apparent change in the blood pressure, and a higher elevation of ICP ensued. Heart rates decreased with increase in ICP, while the rate frequently increased with levels of ICP over about 120 mm Hg. Changes in the vagal and sympathetic discharges always began at a time before the initiation of cardiovascular response to the elevated ICP. However, when ICP was repeatedly increased, the increase in vagal discharges progressively decayed and was accompanied by vigorous sympathetic firings and a marked pressor response. The sympathetic outflow also decayed following the decrease in vagal activities. The present findings of changes in the vagal type 1 discharges demonstrate clear participation of parasympathetic as well as sympathetic nerve activity in the occurrence of cardiovascular responses to increased ICP. Changes in both these autonomic nerve responses may explain the initial fall in arterial blood pressure and pressor responses associated with bradycardia or tachycardia, at different levels of elevated ICP.