Microglia are equipped with a strong proton (H ϩ ) extrusion pathway, a voltage-gated H ϩ channel, probably to compensate for the large amount of H ϩ generated during phagocytosis; however, little is known about how this channel is regulated in pathological states. Because neural damage is often associated with intracellular and extracellular acidosis, we examined the regulatory mechanisms of the H ϩ current of rat spinal microglia in acidic environments. More than 90% of round/amoeboid microglia expressed the H ϩ current, which was characterized by slow activation kinetics, dependencies on both intracellular and extracellular pH, and blockage by Zn 2ϩ . Extracellular lactoacidosis, pH 6.8, induced intracellular acidification and cell swelling. Cell swelling was also induced by intracellular dialysis with acidic pipette solutions, pH 5.5-6.8, at normal extracellular pH 7.3 in the presence of Na ϩ . The H ϩ currents were increased in association with cell swelling as shown by shifts of the half-activation voltage to more negative potentials and by acceleration of the activation kinetics. The acidosis-induced cell swelling and the accompanying potentiation of the H ϩ current required nonhydrolytic actions of intracellular ATP and were inhibited by agents affecting actin filaments (phalloidin and cytochalasin D). The H ϩ current was also potentiated by swelling caused by hypotonic stress. These findings suggest that the H ϩ channel of microglia can be potentiated via cell swelling induced by intracellular acidification. This potentiation might operate as a negative feedback mechanism to protect microglia from cytotoxic acidification and hence acidosis-induced swelling in pathological states of the CNS. Key words: H ϩ channel; lactoacidosis; cell swelling; microglia; pH regulation; ATP; cytochalasin D; cytoskeleton; spinal cordMicroglia are activated in response to various disorders of the CNS, including infection, ischemia, trauma, and neurodegenerative diseases, and they participate in both neuroprotective and neuropathological events (Streit, 1996). They are sensitive to the minor changes in their microenvironment present during very early stages of brain damage, such as subtle imbalances in ion homeostasis, and transform rapidly from the resting to the activated state (Gehrmann et al., 1993;Kreutzberg, 1996). Various ion channels in microglia are considered to contribute to the high responsiveness to pathological events and to be involved in maintenance of the neural microenvironment (Kettenmann et al., 1990;Nörenberg et al., 1994;Schlichter et al., 1996;Eder, 1998).A voltage-gated proton (H ϩ ) channel, first found in snail neurons (Thomas and Meech, 1982), has been suggested to be the mechanism for H ϩ extrusion responsible for compensation of intracellular acidification and for the dissipation of depolarization found in phagocytes that generate a massive amount of H ϩ during respiratory bursts (Lukacs et al., 1993;DeCoursey and Cherny, 1994). Similar H ϩ currents have been described in murine (Eder et al., 19...
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. Introduction: Hϩ is a key signaling ion in bone resorption. In addition to H ϩ pumps and exchangers, osteoclasts are equipped with H ϩ conductive pathways to compensate rapidly for pH imbalance. The H ϩ channel is distinct in its strong H ϩ extrusion ability and voltage-dependent gatings. Methods: To investigate how and when the H ϩ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H ϩ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Results and Conclusions:Whole cell recordings clearly showed that the H ϩ current was enhanced by increasing the pH gradient across the plasma membrane (⌬pH), indicating that the H ϩ channel changed its activity by sensing ⌬pH. The reversal potential (V rev ) was a valuable tool for the real-time monitoring of ⌬pH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 M) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of V rev , suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K
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.
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 (∆pH = 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 A 1 and N ,N -dicyclohexylcarbodiimide. Although H + was transported uphill, the electrochemical gradient for H + affected the current. The currents were increased by elevating ∆pH and depolarization, and were reduced by lowering ∆pH and hyperpolarization. Elevation of extracellular Ca 2+ (5-40 mM) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg 2+ mimicked the inhibition. With 40 mM Ca 2+ , the currents decreased to < 40% at 0 mV and to < 10% at about −80 mV. Increases in the intracellular Ca 2+ (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 Ca 2+ in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.
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