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Osteoclasts secrete protons and various enzymes to degrade bone matrix, and play a crucial role in bodily Ca2+ homeostasis. Both organic and inorganic bone degradation products are transcytosed through osteoclasts and liberated into the extracellular space (Nesbitt & Horton, 1997; Salo et al. 1997). During the bone resorption cycle, osteoclasts face dynamic changes in diverse extracellular/intracellular environmental factors, such as osmolarity, Ca2+ levels, pH, metabolic activities and organization of cytoskeletons (Zaidi et al. 1993; Lakkakorpi & Vaananen, 1996). The ruffled membrane proton secretion via a vacuolar-type H+-ATPase requires anion efflux through the Cl− channel. Resultant accumulation of HCO3− and reduction of intracellular Cl− are compensated for via the HCO3−-Cl− exchanger in the basal membrane (Schlesinger et al. 1994). Transmembrane Cl− flux is thus closely related to the functional state of osteoclasts. Chloride channels have been demonstrated in rat (Sims et al. 1991), avian (Blair & Schlesinger, 1990), rabbit (Kelly et al. 1994) and murine osteoclasts (Shibata et al. 1997), but still little is known about their role in osteoclast functions. So far, the only known activating extracellular stimulus for Cl− channels of osteoclasts is either a rise in the extracellular Ca2+ concentration ([Ca2+]o) (Fujita et al. 1996; Shibata et al. 1997) or hypotonic stress (Kelly et al. 1994). Resorbing activities are inhibited by a rise in [Ca2+]o possibly via a low-affinity receptor-like [Ca2+]o-sensing molecule (Zaidi et al. 1995). Cell swelling induced by hypotonic shock may also lead to changes in various cellular events, such as ion transport through the plasma membrane, organization of the cytoskeleton and metabolic activities (Lang et al. 1998). Osteoclasts would be exposed to both a rise in [Ca2+]o and an osmotic imbalance during the bone resorption. We have shown that a rise in [Ca2+]o activates an outwardly rectifying Cl− (ORCl) channel in in vitro-generated murine osteoclasts (Shibata et al. 1997) which have the characteristics of mature osteoclasts (Akatsu et al. 1992; Shioi et al. 1994), but the hypotonic effects on their Cl− channels remain to be solved. This study aimed at characterizing hypotonically activated Cl− channels in murine osteoclasts and studying the co-operative action of the two stimuli, a rise in [Ca2+]o and osmotic perturbation. We show that the hypotonically activated Cl− current shares common electrophysiological features with the ORCl current activated by a rise in [Ca2+]o, and that a rise in [Ca2+]o shifts the osmolarity set-point for the current activation to respond to a minor change in osmolarity. In addition, we provide evidence that the hypotonically activated ORCl current requires extracellular Ca2+ and intracellular ATP and is inhibited by intracellular alkalization and perturbation of the actin cytoskeleton. A preliminary report has been published (Sakai et al. 1997).

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.

Vacuolar-type H+-ATPases (V-ATPases) are widely distributed at intracellular membranes from yeast to mammalians and could transport H+ across membranes against negative pH gradients. In osteoclasts, V-ATPases are recruited to the plasma membrane by exocytotic fusion of lysozomal membranes to form the ruffled membrane facing to the bone surface and play an important role in acid secretion. Ca2+ released from bone tissue is accumulated in the closed extracellular compartment, which in turn inhibits osteoclast functions. V-ATPases at the ruffled membrane would be exposed to this high level of extracellular Ca2+. We recently succeeded to record proton currents of the plasma membrane V-ATPase in murine osteoclasts using the conventional whole-cell clamp technique, and found that the pump current was decreased by increased extracellular Ca2+. In this study, we investigated whether endocytotic/exocytotic processes were involved in the Ca2+-induced inhibition of the V-ATPase by measuring the H+ current and the cell capacitance (Cm) simultaneously. Cm is a reliable monitor for surface area. Extracellular Ca2+ (5–40 mM) decreased the H+ current and the Cm simultaneously. The Ca2+-induced inhibitions were dose-dependent and were mimicked by Mg2+. The decrease in the Cm by 40 mM Ca2+ was only fractional, corresponding to ∼10% of the control (∼150 pF), but the decrease in the V-ATPase current was by 70–80%. Bafilomycin A1, inhibited the Ca2+-induced decrease in the Cm, indicating that V-ATPase-rich membrane may be targeted for endocytosis. These data suggest that extracellular Ca2+ facilitates internalization of the V-ATPase through endocytosis, which may be a mechanism for Ca2+-induced inhibition of osteoclastic bone resorption.

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 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 ms 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 - 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 high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C, implying that processes with different temperature dependencies underlie the observed Q10 (apparent Q10, Q10app). A novel resistivity pulse method revealed that the access resistance with its low temperature dependence became predominated in high temperature ranges. The Q10app 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 would provide an important clue for the permeation mechanism.