Extracellular nucleotides activate non-selective cation and Ca(2+)-dependent K+ channels in rat osteoclasts.

AUTOR(ES)

Weidema, A F

RESUMO

1. Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2. At negative membrane potentials, ATP (1-100 microM) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations. 3. The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 +/- 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to -21 +/- 3 mV, indicating that ATP activates a non-selective cation current, consistent with involvement of P2X receptors. 4. The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I-V relationship, and reversed at -71 +/- 4 mV. The reversal potential shifted 51 mV per 10-fold change of [K+]o, indicating that ATP activates a K+ current (IK). 5. In fura-2-loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca(2+)-free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br-A23187) to elevate [Ca2+]i, IK activated when [Ca2+]i exceeded approximately 400 nM, with half-maximal activation at 580 +/- 50 nM. 6. In cell-attached patches, ATP activated a channel with a conductance of 48 +/- 6 pS, that reversed director, near EK. Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel. 7. These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non-selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca(2+)-dependent K+ channel.

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