High‐conductance anion channels in embryonic chick osteogenic cells

Patch‐clamp measurements done on excised membrane patches obtained from 1‐5 day cultured embryonic chick osteoblasts, osteocytes, and periosteal fibroblasts revealed the existence of a high‐conductance anion channel: 371 ± 63 pS when measured under symmetrical 158 mM CI− conditions. The channel frequently displayed subconductance levels. The ion selectivity of the channel expressed as the (an)ion to chloride permeability ratio was as follows: CI− (1.0) > methylsulfate− (0.71) > gluconate− (0.25) > glutamate− (0.17) > Na+ = K+ (0.10). In addition, the channel had a significant permeability for inorganic phosphate ions. The channel was found in about 1% of the cell‐attached patches, which indicates that the channel is under the control of as yet unknown intracellular factors. Once activated by patch excision, the channel was voltage dependent and active at potentials close to 0 mV. At potentials outside the range of ± 10 mV channel activity decreased. This process proceeded faster at increasing membrane potentials of either polarity. Returning to potentials close to 0 mV caused reopening of the channels within seconds if the preceding voltage step led to complete closure of the channels. Channel activity did not depend noticeably on intracellular and extracellular Ca2+ ions. The channel is not unique to (chick) osteogenic cells but has been demonstrated in excised patches obtained from excitable and other nonexcitable cells. Although its presence in a wide variety of cell types suggests that the channel plays a general role in as yet unknown cell physiologic processes, the channel may also have specific functions in osteogenic cells, for example providing a pathway for phosphate ions during mineralization

Identification of Ca2+‐activated K+ channels in cells of embryonic chick osteoblast cultures

Primary cultures of embryonic chick osteoblasts consist of a heterogeneous cell population. Patch clamp measurements were done on 1‐ to 5‐day‐old osteoblasts, osteocytes, fibroblastlike cells, and cells that could not be classified on morphologic criteria. The measurements showed the omnipresence of depolarization‐activated high‐conductance channels in cell‐attached patches. The whole‐cell experiments showed an outward rectifying conductance activating at positive membrane potentials. Channels underlying the latter conductance were found to be K+ conducting in outside‐out membrane patches. The activation potential of the outward rectifying K+ conductance shifted to negative membrane potentials upon increasing the intracellular Ca2+ concentration within the range of 10−8–10−3.2 M. The same happened with the activation potential of the K+ channels found in outside‐out patches. Finally, inside‐out patch experiments directly demonstrated the dependency of the activation potential of K+ channels on Ca2+ ions. Thus the identity and main characteristics of Ca2+‐activated K+ channels expressed by the various cell types present in chick osteoblast cultures have now been established. Decreased input resistances were found in cells of cultures more than 2 days old. This is consistent with the establishment of electrical coupling between the cells. Functions in which Ca2+‐activated K+ channels could play a role are discussed

A Ca2+-dependent K+-channel in freshly isolated and cultured chick osteoclasts

Calcium-activated potassium channels were found in embryonic chick osteoclasts using the patch-clamp technique. The activity of the channel was increased by both membrane depolarisation and an increase in intracellular Ca2+ concentration in the range 10-5 to 10-3 M. In the cell-attached-patch configuration the channel was only active at extreme depolarising potentials. Ca2+ addition to the cytoplasm via ionomycin increased channel activity at the resting membrane potential of the osteoclast. The channel had a single-channel conductance of 150 pS in the inside-out patch under symmetrical K+ conditions (150 mM) and was selective for potassium ions. During sustained application of increased [Ca2+] at the cytoplasmic side of inside-out patches, channel activity sometimes decreased again after the initial increase (desensitization). The results established the properties of the single channels underlying an outward rectifying K+ conductance in chick osteoclasts described previously by us

Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells

Embryonic chick bone cells express various types of ionic channels in their plasma membranes for as yet unresolved functions. Chick osteoclasts (OCL) have the richest spectrum of channel types. Specific for OCL is a K+ channel, which activates (opens) when the inside negative membrane potential (Vm) becomes more negative (hyperpolarization). This is consistent with findings of others on rat OCL. The membrane conductance constituted by these channels is called the inward rectifying K+ conductance (GKi), or inward rectifier, because the hyperpolarization‐activated channels cause cell‐inward K+ current to pass more easily through the membrane than outward K+ current. Besides GKi, channels, OCL may express two other types of voltage‐activated K+ channels. One constitutes the transient outward rectifying K+ conductance (GKto), which is activated upon making the membrane potential less negative (depolarization) but has a transient nature. This conductance favors transient K+ conduction in the cell‐outward direction. The GKto also occurs in a small percentage of cells in osteoblast (OBL) and periosteal fibroblast (PFB) cultures. The other OCL K+ conductance, the GKCa, is activated by both membrane depolarization and a rise in [Ca2+]i. GKCa channels are also present in the other chick bone cell types, that is, OBL, osteocytes (OCY), and PFB. Furthermore, in excised patches of all bone cell types, channels have been found that conduct anions, including CI− and phosphate ions. These channels are only active around Vm = 0 mV. While searching for a membrane mechanism for adaptation of bone to mechanical loading, we found stretch‐activated channels in chick osteoclasts; other investigators have found stretch‐activated cation channels (K+ or aselective) in rat and human osteogenic cell lines. In contrast to other studies on cell lines or OBL from other species, we have not found any of the classic macroscopic voltage‐activated calcium conductances (GCa) in any of the chick bone cells under our experimental conditions. However, our fluorescence measurements of [Ca2+]i in single cells indicate the presence of Ca2+ conductive pathways through the plasma membrane of osteoblastic cells and osteoclasts, consistent with other studies. We discuss possible roles for GKi, GKCa, and anion channels in acid secretion by OCL and for stretch‐activated channels in OCL locomotion