Extracellular Nucleotides Activate non-Selective Cation and Ca2+-Dependent K+ Channels in Rat Osteoclasts
Author's email addess
A. F. Weidema: firstname.lastname@example.org
- 1Extracellular 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.
- 2At negative membrane potentials, ATP (1–100 μm) 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.
- 3The 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.
- 4The 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).
- 5In fura-2-loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca2+-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-A23l87) to elevate [Ca2+]iIK activated when [Ca2+]i exceeded ∼400 nm, with half-maximal activation at 580 ± 50 nM.
- 6In cell-attached patches, ATP activated a channel with a conductance of 48 ± 6 pS, that reversed direction near EK Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel.
- 7These 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 Ca2+-dependent K+ channel.