Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺ concentrations ([Na⁺]_i) in MPG neurons. The recovery of [Na⁺]_i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na⁺/K⁺ ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% (n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca²⁺]_i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca²⁺-activated K⁺ channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na⁺]_i and [Ca²⁺]_i, which activates Na⁺/K⁺ ATPase and Ca²⁺-activated K⁺ channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.     

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca²⁺ and voltage-gated potassium (BK_Ca) channels. Here, we explored the effect of LPI on intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels using excised inside-out patches from endothelial cells. The functional expression of IK_Ca was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK_Ca channels with a slope conductance of 39 pS in symmetric K⁺ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK_Ca single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK_Ca single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.     

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 stretche-voked 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, Ca²⁺-activated K⁺ (K_Ca) 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 K_Ca 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 K_Ca 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.     

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A

We identified voltage-activated K⁺ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca²⁺-activated K⁺ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 ± 1.7 pS channels (K_DR1), 90.6 ± 5.4 pS channels (K_DR2) and 149 ± 4 pS intermediate-conductance Ca²⁺-activated K⁺ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from K_DR1 channels inactivated with a time constant of 1.7 ± 0.1 s at +60 mV test potential, while K_DR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca²⁺] (10⁻⁶M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). K_DR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1–10 mM) but not to Ca²⁺. K_DR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of K_DR1 channels 3.4-fold and of K_DR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (−60 mV) only openings of K_DR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K⁺ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells. Received: 20 June 1995/Received after revision: 25 January 1996/Accepted: 7 February 1996     

Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes

Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO₂ equal to 0 mmHg was realized by inclusion of Oxyrase. [Ca²⁺]_i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in [Ca²⁺]_i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic [Ca²⁺ _i were 52±3 nM (N=42) and 2115±59 nM (N=45), respectively. The purported Na⁺ overload blocker R 56865, significantly reduced maximal anoxic [Ca²⁺]_i to 553±56 nM (P<0.05), implicating a role of elevated intracellular Na⁺ in the anoxia-induced increase in [Ca²⁺]_i. Veratridine (30 M), which induces Na⁺ overload, increased [Ca²⁺]_i to 787±39 nM. The compound R 56865 reduced veratridine-induced increases in [Ca²⁺]_i to 152±38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, [Ca²⁺]_i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, [Ca²⁺]_i increased further followed by hypercontraction and loss of cell viability. The mean value for [Ca²⁺]_i 10 min after reperfusion of the former group, was 752±46 nM (N=38). The cardiomyocyte cell shape could be followed by monitoring changes in the total fura-2 fluorescence (340+380 nm signal). Within 15 min after the onset of anoxia, the total fluorescence signal increased suddenly, before [Ca²⁺]_i started to rise, coinciding with the onset of rigor contraction induced by ATP depletion.     

Activation of K+ and Cl− channels by Ca2+ and cyclic AMP in dissociated kidney epithelial (MDCK) cells

In dissociated MDCK cells, activators of the cyclic AMP system cause depolarization detectable by changes in fluorescence of the membrane potential sensitive dye bisoxonol. Addition of forskolin (60 M), vasopressin (2 M), 8-bromo-cyclic AMP (0.5 mM) or l-epinephrine (10 M) depolarized the cells substantially in low Cl^– (5 mM) but had little effect in high Cl^– (140 mM) solution. These results are consistent with cyclic AMP activation of Cl^– channels. The Ca²⁺-ionophore ionomycin (1 M) produced a rapid hyperpolarization in low and high Cl^– solutions, consistent with K⁺ channel opening. Using a clonal subline, MDCK-14, the magnitude of the ionomycin hyperpolarization was roughly proportional to the concomitant rise in [Ca²⁺]_i as measured with the intracellular Ca²⁺ probe indo-I. Both l-epinephrine and isoproterenol appeared to activate the Cl^– channels. However only l-epinephrine produced a [Ca²⁺]_i rise and a transient hyperpolarization (due to K⁺ channel opening), which preceeded the depolarization due to Cl^– channel opening. The l-epinephrine-induced [Ca²⁺]_i response of the heterogeneous MDCK cell population but not of the clonal subline MDCK-14 was inhibited by removal of extracellular Ca²⁺. In the latter only the slow secondary phase of the [Ca²⁺]_i rise was affected by Ca²⁺ removal. It is concluded that l-epinephrine activates K⁺ and Cl^– channels in a sequential manner in MDCK cells by Ca²⁺ and cAMP signals, presumably via - and -adrenergic receptors located on the same cell.Abbreviations MDCK cells Madin Darby Canine Kidney cells - [Ca²⁺]_i intracellular calcium concentration - [Cl^–]_i intracellular chloride concentration - [Cl^–]_o extracellular chloride concentration - [Na⁺+K⁺]_i intracellular concentration of Na⁺ and K⁺ - [Na⁺+K⁺]_o extracellular concentration of Na⁺ and K⁺ - E_M transmembrane potential - E_Cl chloride equilibrium potential - E_K potassium equilibrium potential - bis-oxonol [bis(1,3-diethylthio-barbiturate)] trimethine oxonol - DMSO dimethylsulfoxide - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethyl ether) N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid - NMG⁺ N-methylglucamine - RPMI medium Rosewell Park Memorial Institute medium     

Biphasic rises in cytosolic free Ca2+ in association with activation of K+ and Cl− conductance during the regulatory volume decrease in cultured human epithelial cells

During exposure to a hypotonic solution (55% osmolarity), cultured human epithelial (Intestine 407) cells exhibit a regulatory volume decrease after osmotic swelling. This process is known to involve parallel activation of volume-regulatory K⁺ and Cl^– conductances. Biphasic increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) were observed by microspectrofluorometry, in fura-2-loaded cells upon hypotonic stress. Electrophysiological studies with Ca²⁺-selective and conventional microelectrodes indicated that a biphasic [Ca²⁺]_i increase was associated with a biphasic hyperpolarization, whereas an interposing [Ca²⁺]_i decrease coincided with a transient depolarization. A Ca²⁺ ionophore, ionomycin, produced a sustained Ca²⁺ increase and a prolonged hyperpolarization which was sensitive to the K⁺ channel blocker, quinine. A subsequent hypotonic challenge gave rise to a depolarization, which was sensitive to a stilbene-derivative Cl^– channel blocker, without inducing further changes in [Ca²⁺]_i. Normal cell volume regulation in a hypo-osmotic medium could take place even in the presence of ionomycin. It is concluded that a biphasic [Ca²⁺]_i increase is closely associated with activation of the volume-regulatory K⁺ conductance, and that the interposing [Ca²⁺]_i decrease is neither a causative factor for activation of the volume-regulatory Cl^– conductance nor a prerequisite for regulatory volume decrease in epithelial cells exposed to a hypotonic solution.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Caffeine enhancement of electrical activity through direct blockade of inward rectifying K+ currents in GH3 rat anterior pituitary cells

Treatment of rat anterior pituitary GH₃ cells with caffeine causes a reversible enhancement of electrical activity superimposed over a depolarization of the plasma membrane potential. Similar results are obtained with theophylline, but not with isobutylmethylxanthine or forskolin. The effects of caffeine are not related to Ca²⁺ liberation from intracellular stores since they are not affected by incubation of the cells with ryanodine or thapsigargin. Furthermore, caffeine-induced hyperpolarization of the membrane is not detectable even in cells in which Ca²⁺ liberation from inositol 1,4,5-trisphosphate-sensitive compartments produces a prominent transient hyperpolarization in response to thyrotropin-releasing hormone. Reductions of Ca²⁺-dependent K⁺ currents caused by partial block of L-type Ca²⁺ channels by caffeine are not sufficient to explain the effects of the xanthine, since the results obtained with caffeine are not mimicked by direct blockade of Ca²⁺ channels with nisoldipine. GH₃ cell inwardly rectifying K⁺ currents are inhibited by caffeine. Studies on the voltage dependence of the caffeine-induced effects indicate a close correlation between alterations of electrical parameters and reported values of steady-state voltage dependence of inactivation of these currents. We conclude that, as previously shown for thyrotropin-releasing hormone, modulation of inwardly rectifying K⁺ currents plays a major role determining the firing rate of GH₃ cells and its enhancement by caffeine.     

Endothelial control of vasodilation: integration of myoendothelial microdomain signalling and modulation by epoxyeicosatrienoic acids

Endothelium-derived epoxyeicosatrienoic acids (EETs) are fatty acid epoxides that play an important role in the control of vascular tone in selected coronary, renal, carotid, cerebral and skeletal muscle arteries. Vasodilation due to endothelium-dependent smooth muscle hyperpolarization (EDH) has been suggested to involve EETs as a transferable endothelium-derived hyperpolarizing factor. However, this activity may also be due to EETs interacting with the components of other primary EDH-mediated vasodilator mechanisms. Indeed, the transfer of hyperpolarization initiated in the endothelium to the adjacent smooth muscle via gap junction connexins occurs separately or synergistically with the release of K⁺ ions at discrete myoendothelial microdomain signalling sites. The net effects of such activity are smooth muscle hyperpolarization, closure of voltage-dependent Ca²⁺ channels, phospholipase C deactivation and vasodilation. The spatially localized and key components of the microdomain signalling complex are the inositol 1,4,5-trisphosphate receptor-mediated endoplasmic reticulum Ca²⁺ store, Ca²⁺-activated K⁺ (K_Ca), transient receptor potential (TRP) and inward-rectifying K⁺ channels, gap junctions and the smooth muscle Na⁺/K⁺-ATPase. Of these, TRP channels and connexins are key endothelial effector targets modulated by EETs. In an integrated manner, endogenous EETs enhance extracellular Ca²⁺ influx (thereby amplifying and prolonging K_Ca-mediated endothelial hyperpolarization) and also facilitate the conduction of this hyperpolarization to spatially remote vessel regions. The contribution of EETs and the receptor and channel subtypes involved in EDH-related microdomain signalling, as a candidate for a universal EDH-mediated vasodilator mechanism, vary with vascular bed, species, development and disease and thus represent potentially selective targets for modulating specific artery function.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Facilitation of Ca2+-activated K+ channels (IKCa1) by mibefradil in B lymphocytes

K⁺ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K⁺ channel (LK_bg) in addition to the voltage-gated K⁺ channel (Kv) and Ca²⁺-activated K⁺ channel current (IKCa1). Mibefradil, a blocker of T-type Ca²⁺ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK_bg current with half inhibitory concentration (IC₅₀), 1.9 and 2.3 μM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca²⁺ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 μM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 μM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1. HY Yoo, H Zheng, and JH Nam contributed equally to this study.     

Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca²⁺]_i) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I₂ in endothelial cells. ATP (3 mol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca²⁺]_i in cultured endothelial cells. [Ca²⁺]_i remained elevated over several minutes. When the cells were depolarized, either by K⁺ (70–90 mmol/l) or by preincubation with the blocker of K⁺ channels tetraethylammonium (3 mmol/l), the initial peak of [Ca²⁺]_i remained unaffected but [Ca²⁺]_i returned significantly faster to resting levels, indicating a reduction in Ca²⁺ influx. In native, freshly isolated endothelial cells, K⁺ abolished increases in [Ca²⁺]_i induced by acetylcholine (3 mol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K⁺ (by 70%), whereas release of prostaglandin I₂ was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 mol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca²⁺]_i (twofold) after washout of the drug. Depolarization with K⁺ decreased thimerosal-induced EDRF release and [Ca²⁺]_i in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 mol/l), an activator of K⁺ channels. BRL 34915 also elicited increases in [Ca²⁺]_i, particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K⁺. We conclude that the initial rise in [Ca²⁺]_i in response to receptor-binding agonists, caused by mobilization of Ca²⁺ from intracellular stores, activates K⁺ channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca²⁺ influx into endothelial cells and is thus an important signal for synthesis and release of EDRF.     

Effect of dexamethasone on voltage-gated K+ channels in Jurkat T-lymphocytes

The voltage-gated K⁺ channel Kv1.3 is an important regulator of lymphocyte function. Activation of lymphocytes is accompanied by stimulation, whereas CD95-induced apoptosis by inhibition, of Kv1.3. The channel serves to maintain cell membrane potential, a prerequisite for signalling through the Ca²⁺ release-activated Ca²⁺ channel I_CRAC. As glucocorticoids are known to regulate lymphocyte function, the present study addressed the effect of dexamethasone on voltage-gated K⁺ channels in Jurkat T-lymphocytes. In whole-cell patch-clamp experiments current families evoked by 200-ms potential steps every 15 s from –70 mV to values from –120 to +100 mV revealed the functional expression of voltage-gated K⁺ channels. Pre-treatment of Jurkat T-lymphocytes for 2–3 h with 1 µM dexamethasone led to a significant decrease of voltage-gated K⁺ currents. Fura-2-fluorescence measurements showed that the readdition of Ca²⁺ to Ca²⁺-depleted cells led to a rapid increase of cytosolic Ca²⁺ activity. This increase of Ca²⁺ activity was blunted by both the K⁺ channel blocker margatoxin (10 nM) and 24 h pre-treatment with dexamethasone (1 µM). In conclusion, dexamethasone inhibits voltage-gated K⁺ channels in Jurkat T-lymphocytes, an effect impeding Ca²⁺ entry through I_CRAC.     

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture

Ca²⁺ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca²⁺ ([Ca²⁺]_i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca²⁺]_i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D600) and felodipine on [Ca²⁺]_i during anoxia. A method was developed whereby [Ca²⁺]_i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O₂ was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca²⁺]_i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca²⁺]_i were 118±2 (n=98) and 662±22 (n=160) nM, respectively. D600 (1 M), but not felodipine (10 M), significantly reduced basal [Ca²⁺]_i in normoxic incubations. During anoxia 1 M and 100 M D 600 significantly decreased anoxic [Ca²⁺]_i levels by 22 and 63% respectively. Felodipine at 10 M was as effective as 1 M D600. Removal of extracellular Ca²⁺ and addition of 0.1 mM La³⁺ completely abolished anoxia-induced increases in [Ca²⁺]_i. We conclude that anoxia induces increases in [Ca²⁺]_i in rabbit PT cells in primary culture, which results from Ca²⁺ influx. Since this Ca²⁺ influx is partially inhibited by low doses of CCBs, Ltype Ca²⁺ channels may be involved.     

DIVALENT CATION MODULATION OF A-TYPE POTASSIUM CHANNELS IN ACUTELY DISSOCIATED CENTRAL NEURONS FROM WIDE-TYPE AND MUTANT DROSOPHILA

Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K⁺ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd²⁺ enhanced the peak inactivating current (I_A) without affecting the delayed component (I_K). The same results were obtained in Ca²⁺-free external solution, and from slo¹ mutation, which eliminates transient Ca²⁺-activated K⁺ current. Micromolar Cd²⁺and Zn²⁺, and millimolar Ca²⁺and Mg²⁺ all shifted the steady-state inactivation curve of I_A without affecting the voltage-dependence of I_A activation, whereas millimolar Cd²⁺markedly affected both the activation and steady-state inactivation curves for I_A. Divalent cations affected I_A with different potency; the sequence was: Zn²⁺ > Cd²⁺ > Ca²⁺ > Mg²⁺. The modulation of I_A by Cd²⁺ was partially inhibited in Sh^M, a null Shaker (one of I_A-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on I_A activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K⁺ channels through specific binding to extracellular sites of the channels.     

The calcium channel antagonist diltiazem effectively blocks two types of potassium channels in the neuronal membranes

Effect of the Ca²⁺-channel antagonist diltiazem on potential-operated Ca²⁺ and K⁺ currents was studied on isolated edible snail neurons by a two-microelectrode patch-clamp technique. Diltiazem in a concentration of 0.1 mM inhibits Ca²⁺ current, high-threshold Ca²⁺-dependent K⁺ current, and Ca²⁺-independent K⁺ current and has no effect on low-threshold K⁺ current and leakage current. It is suggested that therapeutic effect of diltiazem is mediated through blockade of Ca²⁺ and K⁺ channels. Tranlated fromByulleten' Eksperimental'noi biologii i Meditsiny, Vol. 124, No. 9, pp. 271–274. September, 1997     

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca²⁺ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca²⁺ action potentials but has a Ca²⁺ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca²⁺ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca²⁺-activated K⁺ tails currents and during Ca²⁺-activated K⁺ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.     

Effects of extracellular K+ and Ca2+ on membrane potential,contraction and86Rb+ efflux in guinea-pig mesotubarium

The effects of varying extracellular concentrations of K⁺ and Ca²⁺ [K⁺]_o and [Ca²⁺]_o on force development and membrane potential were investigated in the guinea-pig mesotubarium. At [K⁺]_o up to 40 mM, spontaneous action potentials were present, while higher [K⁺]_o gave sustained contractures at a stable membrane potential (−24 to −12 mV for [K⁺]_o from 60 to 120 mM). Tension decreased successively with increasing [K⁺]_o from 30 to 120 mM. The relaxing potency of the dihydropyridine Ca²⁺ antagonist, felodipine, increased as the membrane was depolarized with increasing [K⁺]_o and action potentials ceased. These results are compatible with the existence of Ca²⁺ channels showing voltage-dependent affinity with dihydrophyridines. Increasing [Ca²⁺]_o from 2.5 to 10 mM caused membrane hyperpolarization by about 11 mV and was accompanied by a lower frequency of spontaneous contractions and a longer duration of the relaxation between contractions.⁸⁶Rb⁺ efflux measurements in 60 mM K⁺ in the absence and presence of felodipine revealed a Ca²⁺-dependent component of the voltage-activated efflux. In normal solution (5.9 mM K⁺), efflux in the presence of felodipine was similar to the minimal value during normal spontaneous activity. The results indicate regulation of the permeability of K⁺ channels by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and suggest participation of such channels in the generation of the regularly occurring bursts of action potentials characteristic of spontaneous activity in the mesotubarium.