Na+ dependence of extracellular Ca2+-sensing mechanisms leading to activation of an outwardly rectifying Cl− channel in murine osteoclasts

An elevation in the extracellular Ca2+ concentration ([Ca2+o) is a key signal for bone remodeling by inhibiting the resorbing activity of osteoclasts. The [Ca2+]o-sensing responses include a variety of morphological and functional changes, but the underlying mechanisms are yet to be defined. This study was aimed at investigating the [Ca2+]o-sensing mechanisms leading to the activation of the Cl− channel in murine osteoclasts. A rise in either Ca2+ or Gd3+ activated an outwardly rectifying Cl− (ORcl) channel reversibly and dose-dependently, which was characterized by rapid activation kinetics, little inactivation, and blockage by DIDS. The concentration required for a half-maximal response was estimated to be >20–30 mmol/L for Ca2+. Intracellular dialysis with an ATP-free pipette solution or application of an actin destabilizer, cytochalasin D, decreased the [Ca2+]o-activated ORcl current. Substitution of extracellular Na+ by an impermeable cation, N-methyl- -glucamine+, inhibited the [Ca2+]o-activated ORcl channel, suggesting that the activation depended on extracellular Na+. A blocker for the Na+-Ca2+ exchanger, 2′4′-dichlorobenzamil hydrochloride (DCB), inhibited the [Ca2+]o-activated ORcl channel as well. Although 10 mmol/L Ca2+ activated the ORcl current only slightly at a standard intracellular pH (7.3), decreasing pH by dialyzing cells with an acidic pipette solution (pH 6.6) enhanced the [Ca2+]o-activated ORcl current. This potentiation by cell acidosis was eliminated by amiloride, a blocker for the Na+-H+ exchanger. Zinc ion (0.1 mmol/L) and a polycation, neomycin (0.2 mmol/L), activated the ORcl current at intracellular pH 6.6, whereas the effects of those cations were negligible at intracellular pH 7.3. These results suggest that [Ca2+]o-sensing mechanisms, leading to activation of the ORcl channel in murine osteoclasts, are regulated by ATP and actin cytoskeletal organization, and are sensitized greatly by cell acidosis. Contributions of Na+-dependent transporters in this activating process are examined in the context of a possible intermediate signal of cell swelling caused by Na+ influx.