Extracellular Nucleotides Activate non‐Selective Cation and Ca²⁺‐Dependent K⁺ Channels in Rat Osteoclasts

Abstract

Osteoclasts are the cells responsible for resorption of bone and other mineralized tissues, and participate in physiological processes such as bone remodelling and tooth eruption. These multinucleated cells arise from fusion of mononucleated precursors of the monocyte-macrophage lineage that originate in the bone marrow (Roodman, 1996). Osteoclasts exhibit different phases of activity, with cells alternating between motile and resorbing phases. Motile osteoclasts typically exhibit extensive pseudopods and are flattened in appearance (spread morphology). In contrast, resorbing osteoclasts lack extensive pseudopodia and are more dome shaped (rounded morphology). Resorption is accomplished by acidification of a compartment referred to as the resorption lacuna. Transport of H¤ by an electrogenic H¤-ATPase causes dissolution of the mineral phase of bone, while secreted hydrolytic enzymes digest the organic matrix (Roodman, 1996). Since H¤ transport is electrogenic, ion channels are required to provide pathways to dissipate charge. Previous studies have revealed that mammalian osteoclasts express a number of channel types, including an inwardly rectifying K¤ channel (IRK1), a transient outwardly rectifying K¤ channel (Kv1.3), H¤ channels and Cl¦ channels (Arkett, Dixon & Sims, 1992;Kelly, Dixon & Sims, 1992;Arkett, Dixon, Yang, Sakai, Minkin & Sims, 1994;Yamashita, Ishii, Ogata & Matsumoto, 1994;Kelly, Dixon & Sims, 1994; N ordstrom et al. 1995). The expression of channels is related to the morphology of the osteoclast, with spread osteoclasts exhibiting IRK1 and Cl¦ current, were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2. At negative membrane potentials, ATP (1-100 ìÒ) 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¤]ï to 10 mÒ shifted the reversal potential to −21 ± 3 mV, indicating that ATP activates a nonselective 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.

Osteoclast spreading kinetics are correlated with an oscillatory activation of a calcium-dependent potassium current

“…The KCa channels observed in our study have pharmacological characteristics that are different from classic BK, IK and SK channels described in other cells. They are also different from the KCa previously described in osteoclasts (Weidema et al, 1997), which has a 54 pS unitary conductance and is insensitive to venom toxins (CTX, APA and kaliotoxin).…”

“…Activation of a calcium-dependent potassium current has been observed in osteoclasts in response to [Ca 2+ ]i increase induced by extracellular ATP or by A-23187 calcium ionophore application (Weidema et al, 1997). To test the internal calcium dependence of the current, non-spreading osteoclasts were bathed in control solution and voltage clamped using the protocol described in Fig.…”

“…It was demonstrated that internal application of GTP analogues (GTPγS) induces osteoclast spreading and suppresses the inward rectifying potassium current expressed by these cells (Arkett et al, 1994). Calcium transients were observed spontaneously in osteoclasts (Radding et al, 1999) and were also induced by osteoclast attachment to bone or by external stimuli such as ATP and PAF (platelet-activating factor) (Zheng et al, 1993;Weidema et al, 1997). However, no study has yet investigated how ionic channel activity is linked to calcium handling and membrane movements.…”

The role of purinergic signalling in the musculoskeletal system

“…Early investigations also provided evidence for the presence of P2X receptors, giving rise to non-selective cation currents, and P2Y receptors, mediating Ca 2+ release from intracellular stores (Weidema et al, 1997;Wiebe et al, 1999). Initially it was thought that P2X4 receptors mediated the ATP-activated nonselective cation currents in rabbit osteoclasts (Naemsch et al, 1999).…”

Molecular Physiology of P2X Receptors

“…On the basis of radiation hybrid mapping, they were judged to be Ͻ130 kb apart (46). In fact, the genes are adjacent in the genomes of humans (23,492 bp separating) and mice (26,464 bp separating; chromosome 5). This presumably reflects gene duplication, and P2X 4 and P2X 7 subunits are among the most closely related pairs in amino acid sequences (Figs.…”