Biomedical subjects

D L Ypey

Publications and source records attributed to D L Ypey.

At least 19 recordsLinked to original sources

PubMed2005

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations.

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.

Action Potentials↗

PubMed2005

Inhibition of the A-type K+ channels of dorsal root ganglion neurons by the long-duration anesthetic butamben.

n-Butyl-p-aminobenzoate (BAB; butamben) is a long-duration anesthetic used for the treatment of chronic pain. Epidural administration of BAB is thought to reduce the electrical excitability of dorsal root nociceptor fibers by inhibiting voltage-gated ion channels. To further investigate this mechanism, we examined the effects of BAB on the potassium currents of acutely dissociated neurons from the rat dorsal root ganglion (DRG). These neurons express a rapidly inactivating A-type K(+) current (I(A)) that is resistant to tetraethylammonium (20 mM) but inhibited by 4-aminopyridine (5 mM). At low concentrations, BAB (< or =1 microM) selectively inhibited the I(A) component of DRG K(+) current. The voltage dependence of activation and inactivation, kinetics of recovery from inactivation, and the pharmacology of the DRG I(A) were similar to those of the Kv4 family of K(+) channels. Reverse transcription-polymerase chain reaction was used to establish that the messages encoding for all three of the mammalian Kv4 channel subunits (Kv4.1-Kv4.3) were present in the rat DRG. BAB produced a high-affinity, partial inhibition of heterologously expressed Kv4.2 channels (K(D) = 59 nM) but did not alter the kinetics or voltage sensitivity of gating. Substituting polar threonines for conserved hydrophobic residues of the S6 segment weakened BAB binding but did not alter the voltage-dependent gating of the Kv4.2 channel. At physiological pH, BAB is uncharged, suggesting that hydrophobic interactions may contribute to drug binding. The data support a mechanism in which BAB binds near the narrow cytoplasmic entrance of Kv4 channels and inhibits current by a pore blocking mechanism.

Amino Acid Sequence↗

PubMed2004

Modeling action potential generation and propagation in NRK fibroblasts.

Normal rat kidney (NRK) fibroblasts change their excitability properties through the various stages of cell proliferation. The present mathematical model has been developed to explain excitability of quiescent (serum deprived) NRK cells. It includes as cell membrane components, on the basis of patch-clamp experiments, an inwardly rectifying potassium conductance (G(Kir)), an L-type calcium conductance (G(CaL)), a leak conductance (G(leak)), an intracellular calcium-activated chloride conductance [G(Cl(Ca))], and a gap junctional conductance (G(gj)), coupling neighboring cells in a hexagonal pattern. This membrane model has been extended with simple intracellular calcium dynamics resulting from calcium entry via G(CaL) channels, intracellular buffering, and calcium extrusion. It reproduces excitability of single NRK cells and cell clusters and intercellular action potential (AP) propagation in NRK cell monolayers. Excitation can be evoked by electrical stimulation, external potassium-induced depolarization, or hormone-induced intracellular calcium release. Analysis shows the roles of the various ion channels in the ultralong ( approximately 30 s) NRK cell AP and reveals the particular role of intracellular calcium dynamics in this AP. We support our earlier conclusion that AP generation and propagation may act as a rapid mechanism for the propagation of intracellular calcium waves, thus contributing to fast intercellular calcium signaling. The present model serves as a starting point to further analyze excitability changes during contact inhibition and cell transformation.

Action Potentials↗

PubMed2003

Ionic basis for excitability of normal rat kidney (NRK) fibroblasts.

Ionic membrane conductances of normal rat kidney (NRK) fibroblasts were characterized by whole-cell voltage-clamp experiments on single cells and small cell clusters and their role in action potential firing in these cells and in monolayers was studied in current-clamp experiments. Activation of an L-type calcium conductance (GCaL) is responsible for the initiation of an action potential, a calcium-activated chloride conductance (GCl(Ca)) determines the plateau phase of the action potential, and an inwardly rectifying potassium conductance (GKir) is important for the generation of a resting potential of approximately -70 mV and contributes to action potential depolarization and repolarization. The unique property of the excitability mechanism is that it not only includes voltage-activated conductances (GCaL, GKir) but that the intracellular calcium dynamics is also an essential part of it (via GCl(Ca)). Excitability was found to be an intrinsic property of a fraction (approximately 25%) of the individual cells, and not necessarily dependent on gap junctional coupling of the cells in a monolayer. Electrical coupling of a patched cell to neighbor cells in a small cluster improved the excitability because all small clusters were excitable. Furthermore, cells coupled in a confluent monolayer produced broader action potentials. Thus, electrical coupling in NRK cells does not merely serve passive conduction of stereotyped action potentials, but also seems to play a role in shaping the action potential.

Animals↗

PubMed2003

Kv1.1 channels of dorsal root ganglion neurons are inhibited by n-butyl-p-aminobenzoate, a promising anesthetic for the treatment of chronic pain.

In this study, we investigated the effects of the local anesthetic n-butyl-p-aminobenzoate (BAB) on the delayed rectifier potassium current of cultured dorsal root ganglion (DRG) neurons using the patch-clamp technique. The majority of the K(+) current of small DRG neurons rapidly activates and slowly inactivates at depolarized voltages. BAB inhibited the whole-cell K(+) current of these neurons with an IC(50) value of 228 microM. Dendrotoxin K (DTX(K)), a specific inhibitor of Kv1.1, reduced the DRG K(+) current at +20 mV by 34%, consistent with an important contribution of channels incorporating the Kv1.1 subunit to the delayed rectifier current. To further investigate the mechanism of BAB inhibition, we examined its effect on Kv1.1 channels heterologously expressed in mammalian tsA201 cells. BAB inhibits the Kv1.1 channels with an IC(50) value of 238 microM, similar to what was observed for the native DRG current. BAB accelerates the opening and closing of Kv1.1, but does not alter the midpoint of steady-state activation. BAB seems to inhibit Kv1.1 by stabilizing closed conformations of the channel. Coexpression with the Kv beta 1 subunit induces rapid inactivation and reduces the BAB sensitivity of Kv1.1. Comparison of the heterologously expressed Kv1.1 and native DRG currents indicates that the Kv beta 1 subunit does not modulate the gating of the DTX(K)-sensitive Kv1.1 channels of DRG neurons. Inhibition of the delayed rectifier current of these neurons may contribute to the long-duration anesthesia attained during the epidural administration of BAB.

Action Potentials↗

PubMed2002

Sauvagine regulates Ca2+ oscillations and electrical membrane activity of melanotrope cells of Xenopus laevis.

Ca2+ oscillations regulate secretion of the hormone alpha-melanphore-stimulating hormone (alpha-MSH) by the neuroendocrine pituitary melanotrope cells of the amphibian Xenopus laevis. These Ca2+ oscillations are built up by discrete increments in the intracellular Ca2+ concentration, the Ca2+ steps, which are generated by electrical membrane bursting firing activity. It has been demonstrated that the patterns of Ca2+ oscillations and kinetics of the Ca2+ steps can be modulated by changing the degree of intracellular Ca2+ buffering. We hypothesized that neurotransmitters known to regulate alpha-MSH secretion also modulate the pattern of Ca2+ oscillations and related electrical membrane activity. In this study, we tested this hypothesis for the secretagogue sauvagine. Using high temporal-resolution Ca2+ imaging, we show that sauvagine modulated the pattern of Ca2+ signalling by increasing the frequency of Ca2+ oscillations and inducing a broadening of the oscillations through its effect on various Ca2+ step parameters. Second, we demonstrate that sauvagine caused a small but significant decrease in K+ currents measured in the whole-cell voltage-clamp, whereas Ca2+ currents remained unchanged. Third, in the cell-attached patch-clamp mode, a stimulatory effect of sauvagine on action current firing was observed. Moreover, sauvagine changed the shape of individual action currents. These results support the hypothesis that the secretagogue sauvagine stimulates the frequency of Ca2+ oscillations in Xenopus melanotropes by altering Ca2+ step parameters, an action that likely is evoked by an inhibition of K+ currents.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

A slow outward current and a hypoosmolality induced anion conductance in embryonic chicken osteoclasts.

In this paper we report on a hypoosmolality induced current, I(osmo), in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward current, I(slo). I(slo) was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and activated at membrane potentials more positive than -44 +/- 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba2+ (1 mM), blockers of K+ conductances in chicken osteoclasts, did not influence I(slo). However, I(slo) was inhibited by 50 microM extracellular verapamil, which allowed us to study I(osmo) in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization activated I(osmo). It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 +/- 0.9 min (n = 9). The current activated within a few ms upon depolarization and did not inactivate during at least 5 sec. I(osmo) reversed around the calculated Nernst potential for Cl- (E(Cl) = +7.3 mV and V(rev) = +5.4 +/- 3.6 mV, n = 9). The underlying conductance, G(osmo) exhibited moderate outward rectification around 0 mV in symmetrical Cl- solutions. Ion substitution experiments showed that G(osmo) is an anion conductance with P(Cl) approximately = P(F) > P(gluc) >> P(Na). I(osmo) was blocked by 0.5 mM SITS but 50 microM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba2+, 50 microM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl- currents have been implicated in charge neutralization during osteoclastic acid secretion for bone resorption. The present results imply that osmolality may be a factor controlling this charge neutralization.

Animals↗

PubMed2001

Fenamates: a novel class of reversible gap junction blockers.

The effect of fenamates on gap junctional intercellular communication was investigated in monolayers of normal rat kidney (NRK) fibroblasts and of SKHep1 cells overexpressing the gap junction protein connexin43 (Cx43). Using two different methods to study gap junctional intercellular communication, single electrode voltage-clamp step response measurements and dye microinjection, we show that fenamates are reversible blockers of Cx43-mediated intercellular communication. After adding fenamates to a confluent monolayer of electrically coupled NRK fibroblasts, the voltage step-induced capacitive current transient changed from a transient characteristic for charging multiple coupled cell capacitances to one characteristic for a single cell in isolation. The capacitance of completely uncoupled cells was 19.7 +/- 1.0 pF (mean +/- S.E.M.; n = 11). Junctional conductance between the patched cell and the surrounding cells in the monolayer changed from >140.7 +/- 9.6 nS (mean +/- S.E.M.; n = 14) to <1.4 +/- 0.4 nS (mean +/- S.E.M.; n = 11) after uncoupling. Electrical coupling could be restored to >51.8 +/- 4.2 nS (mean +/- S.E.M.; n = 11) by washout of the fenamates. Voltage-clamp step response measurements showed that the potency of fenamates in inhibiting electrical coupling decreases in the order meclofenamic acid > niflumic acid > flufenamic acid. The half-maximal concentration determined by dye-coupling experiments was 25 and 40 microM for meclofenamic acid and flufenamic acid, respectively. Inhibition of gap junctional communication by fenamates did not involve changes in intracellular calcium or pH, and was unrelated to protein kinase C activity or an inhibition of cyclooxygenase activity. Voltage-clamp step response measurements in confluent monolayers of SKHep1 cells that had been stably transfected with Cx43 revealed that fenamates are potent blockers of Cx43-mediated intercellular communication. In conclusion, fenamates represent a novel class of reversible gap junction blockers that can be used to study the role of Cx43-mediated gap junctional intercellular communication in biological processes.

Cell Communication↗

PubMed1999

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system.

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue.

Action Potentials↗

PubMed1997

Hyperpolarization-activated currents in the growth cone and soma of neonatal rat dorsal root ganglion neurons in culture.

Dissociated dorsal root ganglion neuron growth cones and somata from neonatal rats were voltage and current clamped with the use of the perforated-patch whole cell configuration to study the occurrence and properties of slow hyperpolarization-activated currents (Ih) at both regions. Under voltage-clamp conditions Ih, blockable by 2 mM extracellular CsCl, was present in 33% of the growth cones tested. Its steady-state activation as a function of voltage could be fitted with a single Boltzmann function with a midpoint potential of -97 mV. The time course of current activation could be best described by a double-exponential function. The magnitude of the fully activated conductance was 3.5 nS and the reversal potential amounted to -29 mV. At the soma, Ih was found in 80% of the somata tested, which is much higher than occurrence at the growth cone. The steady-state activation curve of Ih at the soma, fitted with a single Boltzmann function, had a midpoint potential of -92 mV, which was more positive than that in the growth cone. The double-exponential activation of the current was faster than in the growth cone. The fully activated conductance of 5.1 nS and the reversal potential of -27 mV were not significantly different from the values obtained at the growth cone. Membrane hyperpolarization by current-clamp pulses elicited depolarizing sags in 30% and 78% of the tested growth cones and somata, respectively, which is in agreement with our voltage-clamp findings. Termination of the hyperpolarizing current pulse evoked a transient membrane depolarization or an action potential at both sites. Application of 2 mM extracellular CsCl hyperpolarized the membrane potential reversibly by approximately 5 mV and blocked the depolarizing sags and action potentials following the current injections at these regions. Thus Ih contributes to the resting membrane potential and modulates the excitability of both the growth cone and the soma. Intracellular perfusion with the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) was only possible at the soma by the use of the conventional whole cell configuration. Addition of 100 microM cAMP to the pipette solution shifted the midpoint potential of the Ih activation curve from -108 to -78 mV. The current activation time course was also accelerated. The reversal potential and the fully activated conductance underlying Ih were not changed by cAMP. These results imply that cAMP primarily affects the gating kinetics of Ih. Our results show for the first time quantitative differences in Ih properties and occurrence at the growth cone and soma membrane. These differences may reflect differences in intracellular cAMP concentration and in the expression of Ih.

Animals↗

PubMed1995

Tetrodotoxin-sensitive fast Na+ current in embryonic chicken osteoclasts.

A voltage-dependent, fast, transient inward current was characterized in embryonic chicken osteoclasts using the permeabilized patch configuration of the patch-clamp technique. The current was activated by depolarizations to higher than -28 +/- 4 mV from a holding potential of -80 mV. It peaked within 1-1.5 ms, and inactivated within 3.3-6.9 ms. The 50% inactivation voltage was -59 +/- 6 mV with a steepness factor of 0.11 +/- 0.06. The current disappeared with the removal of extracellular Na+ and was reversibly blocked by tetrodotoxin (K0.5 < 15 nM) but not by verapamil (< or = 100 microM). We conclude that this new current in embryonic chicken osteoclasts is a sodium current known from excitable cells.

Animals↗

PubMed1995

Cell membrane stretch in osteoclasts triggers a self-reinforcing Ca2+ entry pathway.

Many cell types respond to mechanical membrane perturbation with intracellular Ca2+ responses. Stretch-activated (SA) ion channels may be involved in such responses. We studied the occurrence as well as the underlying mechanisms of cell membrane stretch-evoked responses in fetal chicken osteoclasts using separate and simultaneous patch-clamp and Ca2+ imaging measurements. In the present paper, evidence is presented showing that such responses involve a self-reinforcing mechanism including SA channel activity, Ca(2+)-activated K+ (KCa) channel activity, membrane potential changes and local and general intracellular Ca2+ ([Ca2+]i) increases. The model we propose is that during membrane stretch, both SA channels and KCa channels open at membrane potential values near the resting membrane potential. SA channel characterization showed that these SA channels are permeable to Ca2+. During membrane stretch, Ca2+ influx through SA channels and hyperpolarization due to KCa channel activity serve as positive feedback, leading ultimately to a Ca2+ wave and cell membrane hyperpolarization. This self-reinforcing mechanism is turned off upon SA channel closure after cessation of membrane stretch. We suggest that this Ca2+ entry mechanism plays a role in regulation of osteoclast activity.

Animals↗

PubMed1994

Differential depolarization-activated calcium responses in fetal and neonatal rat osteoblast-like cells.

The present study evaluates differential occurrence of voltage-dependent calcium channels (VDCC) in the membranes of fetal (FROB) and neonatal (NROB) calvarian rat osteoblastic cells in primary culture. The intracellular calcium concentration ([Ca2+]i) was monitored upon depolarization of the cell membrane with the use of high K+ containing extracellular solutions. [Ca2+]i was measured in populations of cells as well as in individual cells using Fura-2, whereas the membrane potential (Em) was recorded in parallel experiments using patch-clamp techniques. Increasing the extracellular K+ concentration resulted in an instantaneous depolarization of Em of both FROB and NROB. This depolarization of Em did not significantly affect [Ca2+]i of populations of FROB and neonatal osteoblast precursors (NpROB). In contrast to FROB and NpROB, NROB populations responded to depolarization with significant transient [Ca2+]i increases that could be blocked by the calcium antagonist verapamil and were absent if extracellular Na+ was replaced for choline instead of K+. In individual cell measurements, response frequencies as well as the magnitude of [Ca2+]i responses upon depolarization of NROB were much higher than those of FROB, suggesting that more NROB than FROB possess VDCC. This phenomenon might point to a development-related expression of VDCC in the membranes of osteoblast-like cells.

Animals↗

PubMed1994

Resting membrane potentials and excitability at different regions of rat dorsal root ganglion neurons in culture.

To study the role of electrical membrane processes in neuronal regeneration and growth, resting membrane potentials and action potentials of sensory (dorsal root ganglion) neurons growing in culture were measured at the soma, neurite and growth cone using the whole-cell patch-clamp technique. Our results show that resting membrane potentials measured at the soma (-56.8 +/- 8.8 mV), neurite varicosity (-55.8 +/- 5.2 mV) and growth cone (-57.2 +/- 4.1 mV) of growing neurons were not statistically different. The membrane resistance measured around the resting membrane potential at the neurite varicosity (160 +/- 70 M omega) was smaller than those at the soma (687 +/- 540 M omega) and growth cone (922 +/- 825 M omega). The resting membrane potential measured at the soma using a perforated patch (-60.3 +/- 4.4 mV) was not different from that measured in the normal whole cell. In both configurations, isotonic KCl (140 mM) depolarized the membrane potential to above 0 mV. The K+ channel blockers quinine, Cs+, 4-aminopyridine and tetraethylammonium depolarized the membrane potential by 10-40 mV, while Na(+)-free extracellular solution hyperpolarized it by about 10 mV. Extracellularly applied ouabain, intracellular Na(+)-free or low Cl(-)-containing solutions did not affect the resting membrane potential. Similar results were obtained for growth cones. Action potentials could be evoked by current pulses in 81% of somata and in all growth cones, but not in neurite varicosities. Current-induced repetitive firing was found in 19% of somata and in 65% of growth cones.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1994

Analysis of decaying unitary currents in on-cell patches of cells with a high membrane resistance.

Under nonideal voltage-clamp conditions, unitary currents in the cell-attached patch (CAP) configuration of the patch-clamp technique are decaying instead of rectangular. From these data, four parameters can be determined when multiple identical channels are present. We analyzed an electrical equivalent circuit of the CAP configuration. This analysis yielded, for single-channel resistance, attached-cell membrane resistance, patch resistance, and attached-cell membrane capacitance, a set of different equations to calculate their values from these four parameters. To choose the most robust equation, those that yielded the same resistor/capacitance were compared on the basis of their sensitivity for inevitable deviations of the parameters from their nominal values. The results of our theoretical study are of methodological interest: 1) because they confirm that, under special conditions, CAP measurements can be used to measure electrical membrane properties of intact cells (this is important inasmuch as the CAP is the only configuration in which the cytoplasm of cells remains undisturbed) and 2) because we identify the level of voltage clamp and the equations with which the electrical properties can be determined with optimal accuracy.

Animals↗

PubMed1994

Ionic currents during action potentials in mammalian skeletal muscle fibers analyzed with loose patch clamp.

The loose patch-clamp technique was applied to analyze transmembrane currents during propagating action potentials in superficial fibers of musculi extensor digitorum longus of the mouse in vitro. Experimentally three components were identified in the transmembrane current: 1) a capacitive, 2) an inward sodium, and 3) an outward potassium current. Other components were negligible. The capacitive current was similar in shape to the first derivative of the intracellularly measured action potential. Tetrodotoxin, tetraethylammonium, and 4-aminopyridine, applied in the pipette, were used to identify the contribution in the current by sodium and potassium ions. With extracellularly applied depolarization steps only a sodium current was observed, not a potassium current. Occasionally found outward currents were artifactual. The behaviour of delayed rectifier potassium channels in muscle fiber membranes is discussed in the light of these unexpected findings. We conclude that potassium channel activity contributing to and measured during action potential generation is in some way inaccessible to loose patch extracellular voltage-clamp stimulation and that loose patch action current recording is a useful noninvasive method to analyze membrane conductances involved in action potential generation.

4-Aminopyridine↗

PubMed1994

Effects of bretylium tosylate on voltage-gated potassium channels in human T lymphocytes.

Using the patch-clamp technique, we determined that bretylium tosylate, a quaternary ammonium compound possessing immunomodulating activity, decreased the whole-cell K+ current in human T lymphocytes, in a dose-dependent manner, in the 0.05-5 mM extracellular concentration range. Bretylium tosylate prolonged the recovery from inactivation and accelerated the inactivation and deactivation of the K+ current but did not influence the kinetics of activation or the voltage dependence of activation and steady state inactivation of the K+ conductance. The percentage of drug-induced block was independent of membrane potential. K+ channel block by bretylium tosylate was partially and slowly removable by washing with drug-free extracellular solution. Bovine serum albumin (10 mg/ml) in the bath lifted the drug-induced block almost instantaneously, although not completely. In control experiments bovine serum albumin increased the inactivation time constant of the K+ channels but left the peak K+ current amplitude unaffected. On the basis of the experimental evidence, a gating-dependent allosteric interaction is suggested for the mechanism of drug action. The effective dose range, time of exposure, and reversibility of bretylium tosylate-induced K+ channel block correlated well with the same parameters of the drug-induced inhibition of T lymphocyte activation. The reported effects of bretylium tosylate on T cell mitogenesis can be regarded partly as a consequence of its blocking effects on voltage-gated K+ channels.

Bretylium Tosylate↗

PubMed1993

A Ca(2+)-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 increases (desensitization). The results established the properties of the single channels underlying an outward rectifying K+ conductance in chick osteoclasts described previously by us.

Animals↗