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During resorption of mineralized tissues, osteoclasts are exposed to marked changes in the concentration of extracellular Ca2+ and H+. We examined the effects of these cations on two types of K+ currents previously described in these cells. Whole-cell patch clamp recordings of membrane currents were made from osteoclasts freshly isolated from neonatal rats. In control saline (1 mM Ca2+, pH 7.4), the voltage-gated, outwardly rectifying K+ current activates at approximately -45 mV and the conductance is half-maximally activated at -29 mV (V0.5). Increasing [Ca2+]out rapidly and reversibly shifted the current-voltage (I-V) relation to more positive potentials. Current at -29 mV decreased to 28 and 9% of control current at 5 and 10 mM [Ca2+]out, respectively. This effect of elevating [Ca2+]out was due to a positive shift of the K+ channel voltage activation range. Zn2+ or Ni2+ (5 to 500 microM) also shifted the I-V relation to more positive potentials and had additional effects consistent with blockade of the K+ channel. Based on the extent to which these divalent cations affected the voltage activation range of the outwardly rectifying K+ current, the potency sequence was Zn2+ > Ni2+ > Ca2+. Lowering or raising extracellular pH also caused shifts of the voltage activation range to more positive or negative potentials, respectively. In contrast to their effects on the outwardly rectifying K+ current, changes in the concentration of extracellular H+ or Ca2+ did not shift the voltage activation range of the inwardly rectifying K+ current.(ABSTRACT TRUNCATED AT 250 WORDS)

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Osteoclasts are the cells responsible for the resorption of bone and other mineralized tissues. GTP-binding proteins (G proteins) play important roles in regulating the activity of many cell types; however, there is limited knowledge of their functions in osteoclasts. We used the patch-clamp technique in the whole-cell configuration to introduce either hydrolysis-resistant guanosine triphosphate analogues or fluoroaluminate into single rat osteoclasts, and examined the effects of G protein activation on cell morphology and ionic conductances. Guanosine 5'-O-(3-thiotriphosphate) or 5'-guanylyl-imidodiphosphate, but not the control compounds adenosine 5'-O-(3-thiotriphosphate) or guanosine 5'-O-(2-thiodiphosphate), induced: (1) prompt spreading due to extension of lamellipodia; and (2) after a latency of several minutes, complete suppression of the inwardly rectifying K+ current. Pertussis toxin did not alter either spreading or suppression of K+ current induced by guanosine 5'-O-(3-thiotriphosphate). Cytochalasin D, but not colchicine, prevented guanosine 5'-O-(3-thiotriphosphate)-induced spreading, consistent with actin polymerization underlying lamellipod extension. Whole-cell capacitance did not change during guanosine 5'-O-(3-thiotriphosphate)-induced spreading, which is consistent with a lack of change in total plasma membrane area. Fluoroaluminate did not induce spreading, but it did suppress the K+ current. The differential effects of fluoroaluminate and guanosine 5'-O-(3-thiotriphosphate) suggest that lamellipod extension is regulated by a small molecular mass, monomeric G protein, whereas the inwardly rectifying K+ current is regulated by a large molecular mass, heterotrimeric G protein. Thus, osteoclast motility and ion transport are regulated by separate G protein-coupled pathways.

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Previous studies have revealed that expression of K+ channels in osteoclasts correlates with cell morphology and is influenced by interaction with the extracellular matrix. In this study, we investigated the electrophysiological properties of an outwardly rectifying K+ channel in rat and mouse osteoclasts using patch-clamp techniques. Cell-attached patch recordings revealed a channel of approximately 14 pS conductance that opened upon depolarization, and had a reversal potential close to that predicted for a K+ channel. Channel activity was transient; inactivation of ensemble currents, like that of whole-cell currents, occurred as a single exponential process. Both single-channel and macroscopic currents exhibited use-dependent inactivation in response to repetitive depolarizations. Two scorpion toxins, margatoxin and charybdotoxin, blocked this transient K+ channel, with half-maximal inhibition at 200 pM and 5 nM, respectively. In contrast, dendrotoxin (500 nM) had little effect. In summary, the outwardly rectifying K+ channel in osteoclasts resembles the Shaker-related K+ channel, Kv1.3. When membrane potential was recorded in whole-cell configuration, charybdotoxin (50 nM) caused a depolarization of 5 to 10 mV from resting levels of -50 mV or more positive; therefore this K+ channel contributes to the membrane potential of osteoclasts under some conditions. To investigate the molecular nature of osteoclast K+ channels, we performed RT-PCR on osteoclast RNA using primers for Kv1.3 and the inward rectifier, IRK1. mRNA encoded by Kv1.3 and IRK1 was detected and message identity confirmed by restriction enzyme digestion and sequence analysis. We conclude that osteoclasts exhibit, in addition to the previously described inward rectifier, an outwardly rectifying K+ conductance with properties of the Kv1.3. channel.

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1. We studied the electrophysiological properties of freshly isolated rat osteoclasts using the whole-cell configuration of the patch-clamp technique. Membrane currents were recorded from cells plated on three substates: dentine, type I collagen and glass. 2. Based on their morphology, we defined two categories of osteoclasts. 'Rounded' osteoclasts were dome-shaped and lacked lamellipodia. 'Spread' osteoclasts were flattened and had lamellipodia. The proportion of 'rounded' osteoclasts was significantly greater when cells were plated on dentine or type I collagen than when cells were plated on glass. 3. 'Spread' osteoclasts expressed an inwardly rectifying K+ conductance regardless of the substrate on which they were plated. 4. 'Rounded' osteoclasts, on all substrates, expressed a transient, outwardly rectifying conductance that was selective for K+ based on: reversal of deactivation tail currents at -74 mV; a 60 mV shift in tail current reversal potential for 10-fold change in [K+]o; and blockade of outward current by extracellular 4-aminopyridine, charybdotoxin, and intracellular Cs+. The outward K+ current had an activation threshold of approximately -50 mV, with half-activation at -29 mV. The current also exhibited voltage-dependent inactivation, with half-inactivation at approximately -40 mV. 5. Outward K+ current in 'rounded' osteoclasts was reduced when extracellular Ca2+ was removed and upon addition of Ni2+, but was unaffected by Cd2+ or nifedipine. 6. 'Rounded' osteoclasts had large whole-cell capacitance for their apparent surface area. Capacitance was positively correlated with K+ conductance. The additional surface membrane we detected through capacitance measurements may be the 'ruffled border' of actively resorbing osteoclasts. 7. We conclude that substrate influences the expression of osteoclast phenotype, as defined by morphology and K+ conductances. 'Rounded' osteoclasts express an outwardly rectifying K+ conductance, with no apparent inwardly rectifying K+ conductance. In contrast, 'spread' osteoclasts exhibit an inwardly rectifying K+ conductance with no outwardly rectifying K+ conductance. The 'spread' phenotype may represent a motile phase, while the 'rounded' phenotype may represent a resorptive phase of osteoclastic activity.

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The rhythmical ciliary arrest behavior characteristic of the veliger larvae of the prosobranch Calliostoma ligatum develops in a predictable sequence of events. Spontaneous, small-amplitude (1-3 mV) postsynaptic potentials (PSPs) are first recorded intracellularly from prototrochal ciliated cells at about 45 h after fertilization. Prototrochal ciliated cells, which are precursors of the locomotory, preoral ciliated cells of mature veligers, are electrically coupled to each other. Cilia beat continuously and erratically at this stage. PSP amplitude and duration gradually increase with age, and at about 56 h, preoral ciliated cells become electrically excitable. A single regenerative action potential first occurs at this time and causes a velum-wide, ciliary arrest. Between 56 and 72 h, the duration of the depolarizing phase of the preoral ciliated cell action potential decreases, the amplitude increases, and the hyperpolarizing undershoot develops. Preoral ciliated cell action potentials appear to be Ca2+-dependent throughout development. Shortening of the action potential duration and development of the hyperpolarizing undershoot may be due to activation of later developing K+ channels. As veligers become competent to metamorphose, the preoral velar cells and their connections with the body deteriorate.

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Silver impregnations, immunofluorescence microscopy, and electron microscopy of the nervous system of Velella confirm previous reports that there are two nerve nets, one composed of small and the other of "giant" neurites. Only one of these systems, the small-fibered open one, shows FMRFamide-like immunoreactivity. It appears to be primarily a sensory network. Despite presence of a neuropeptide in these neurons, they did not contain dense-cored vesicles. The "giant" nerve net (closed system) shows many connections that appear syncytial in the silver preparations. While it is confirmed that gap junctions are present between some neurites in the closed system, it is likely that fusion of neurites also occurs and that the system is a partial syncytium. Membrane complexes with gap junctions are abundant in the cytoplasm. It is suggested that fusion occurs by the engulfment of small neurons by large, resulting in an excess of cell membrane, which is internalized with gap junctions still intact. These internalized membranes appear to break up into vesicles eventually. A similar process may occur in the "giant" swimming motor neuron net of the medusa Polyorchis.

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Intracellular recordings from pre-oral ciliated cells of competent Calliostoma ligatum veligers were used to demonstrate the mechanisms of neuronal control of ciliary locomotion. During normal ciliary beating at 5-7 Hz, the membrane potential shows no oscillations or spiking activity. It remains at a resting potential of about -60 mV. Depolarization from resting potential is due to excitatory input from the CNS and, depending upon the kind of input, veligers appear to show two types of locomotory behavior. In one type, normal ciliary beating is periodically interrupted by rapid, velum-wide ciliary arrests. These arrests are caused by a propagated, Ca++-dependent action potential in the pre-oral ciliated cells. The second type is characterized by either a velum-wide or local slowing of normal ciliary beating, and appears to result from a slow depolarization of the ciliated cell membrane. Pre-oral ciliated cells are electrically coupled to each other. This property may ensure the synchrony of velum-wide ciliary arrests or differential velar slowing of ciliary beating. These findings demonstrate some of the mechanisms ofthe fine control veligers possess over their locomotory and feeding behavior.