Hiromu Sakai

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).

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.

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.

pH dependence and inhibition by extracellular calcium of proton currents via plasmalemmal vacuolar‐type H+‐ATPase in murine osteoclasts

The vacuolar‐type H+‐ATPase (V‐ATPase) in the plasma membrane of a variety of cells serves as an acid‐secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V‐ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V‐ATPase in murine osteoclast‐like cells and investigated its activity over a wide range of pH gradients across the membrane (ΔpH = extracellular pH – intracellular pH). The V‐ATPase currents were identified as outward H+ currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A1 and N,N′‐dicyclohexylcarbodiimide. Although H+ was transported uphill, the electrochemical gradient for H+ affected the current. The currents were increased by elevating ΔpH and depolarization, and were reduced by lowering ΔpH and hyperpolarization. Elevation of extracellular Ca2+ (5–40 mm) diminished the currents in a dose‐dependent manner and made the voltage dependence more marked. Extracellular Mg2+ mimicked the inhibition. With 40 mm Ca2+, the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca2+ (0.5–5 μm) did not affect the current. The data suggest that acid secretion through the plasmalemmal V‐ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity‐dependent accumulation of acids and Ca2+ in the closed extracellular compartment might serve as negative feedback signals for regulating the V‐ATPase.

Increases in intracellular pH facilitate endocytosis and decrease availability of voltage-gated proton channels in osteoclasts and microglia

•  Voltage‐gated H+ channels help to compensate for the pH and voltage disturbances generated by production of reactive oxygen species. •  In this study, we investigated how changes in the intracellular pH levels control H+ channel activity in macrophage‐lineage cells, osteoclasts and microglia. •  An increase in intracellular pH decreased the numbers of H+ channels available at the plasma membrane through facilitation of dynamin‐dependent endocytic internalization. •  This inhibitory regulation mechanism for H+ channels is novel. •  The results help us to understand better the significance of the intracellular pH levels in membrane dynamics and H+ channel availability, which, in turn, may modulate natural immunity.

Differential effect of high extracellular Ca2+ on K+ and Cl- conductances in murine osteoclasts.

Abstract. Effects of the extracellular Ca2+ concentration ([Ca2+]o) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca2+ was mediated by a Ba2+-sensitive, inwardly rectifying K+ (IRK) current. A rise in [Ca2+]o (5–40 mm) inhibited the IRK current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl− (ORCl) current. The activation of the ORCl current developed slowly and needed higher [Ca2+]o than that required to inhibit the IRK current. The inhibition of the IRK current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the ORCl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca2+ level neither reduced the IRK current nor activated the ORCl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca2+]o-induced changes in the IRK and ORCl conductances. These results suggest that high [Ca2+]o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IRK conductance and an activation of an ORCl conductance. The two conductances modulated by [Ca2+]o may be involved in different phases of bone resorption because they differed in Ca2+ sensitivity, temporal patterns of changes and regulatory mechanisms.

Electrogenicity of Vacuolar H+-ATPases at the Plasma Membrane of Osteoclasts

The vacuolar-type H+-ATPase (V-ATPase) is an electrogenic H+ pump that is distributed widely in living organisms. Electrogenicity contributes to the proton motive force, but its precise evaluation is difficult mostly because V-ATPases are abundant at the intracellular membranes of acidic vesicles where fluxes of counter ions and H+-leakage could not be fully controlled. In osteoclasts, bone-resorbing cells, V-ATPases are recruited to the plasma membrane (the ruffled membrane) by exocytotic fusion of lysozomes. The electogenicity of the plasma membrane V-ATPases was evaluated under the whole-cell current-clamp recordings in the absence of Na+, K+ and Cl-, where H+ was a major determinant of the membrane potentials. Under pHo/pHi 7.3/5.5 (ΔpH 1.8), the membrane potential varied greatly among cells, from −250 - −7 mV. Bafilomycin, a specific blocker for V-ATPases, depolarized cells by several to 220 mV. The depolarization was dependent on the amplitudes of the V-ATPase currents and eliminated by replacing intracellular ATP by ADP. The V-ATPase-mediated potential was reduced by decreasing ΔpH and disappeared at near ΔpH −2.5. As the whole-cell leak conductance was ∼0.23 nS (the area resistance of the plasma membrane: 6 x 105 Ω cm2), 10 pA of V-ATPase currents could yield the membrane potential of ∼ 40 mV. Small ΔpH-dependent potential due to H+ leakage currents remained in the presence of bafilomycin. Potentials generated by V-ATPases and H+ leak are thus identified successfully. The ruffled membrane could provides a useful model for investigating H+ fluxes across the membrane energized by V-ATPases.

123 Electrophysiological and morphological development of cultured spinal neurons

FUSAO NAKAMURA, MIYUKI KUNO, HIROMU SAKAI, HIROKAZU MORIHATA, SHIUSHI MATSUURA We investigated electrophysiological and morphological development of spinal neurons in vitro. The cells were obtained from rat embryo (Wistar rats, E13-14) and were cultured for up to 4 weeks. The area of soma was smaller th& 300 pm2 within 1 week in vitro and the number of larger cells (>300 ,um2) gradually increased. Most cells exhibited voltage-dependent outward and inward conductances in the whole cell clamp configuration within 3 days in vitro. The outward current consists of two components, 4-aminopyridine-sensitive and TEA-sensitive Kf currents. The transient inward current was activated at around -4OmV and was blocked by TTX, suggesting that the current was mediated by Na +. The Na+ currents tended to be larger in later culture period. Glycine (O.Ol-1mM) increased a conductance with the reversal potential at -40 N -5OmV. These results suggest that most spinal cells developed in vitro exhibit voltage-dependent Na+ and K+ channels and respond to glycine.