Migration of transformed renal epithelial cells is regulated by K+ channel modulation of actin cytoskeleton and cell volume

Migration of transformed renal epithelial (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel (IK channel; Pflügers Arch 432:R87–R93, 1996). This study was aimed at elucidating the functional link between the IK channel and the actin cytoskeleton which is required for cell locomotion. We monitored migration of MDCK-F cells with video microscopy, quantified filamentous actin with phalloidin binding, and measured the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with the fluorescent dye fura-2/AM. We compared the effects of IK channel activation or inhibition with those of hypotonic swelling or hypertonic shrinkage. IK channel inhibition with charybdotoxin (CTX) or cell swelling (omission of up to 50 mmol/l NaCl) as well as IK channel activation with 1-ethyl-2-benzimidazolinone (1-EBIO) or cell shrinkage (addition of up to 100 mmol/l mannitol) reduce the rate of migration dose-dependently by up to 80%, i.e., to the same extent as cytochalasin D. Inhibition of migration is accompanied either by actin depolymerization (CTX and cell swelling) or by actin polymerization (1-EBIO and cell shrinkage). Changes of migration and phalloidin binding induced by CTX and cell swelling or by 1-EBIO and cell shrinkage, respectively, are linearly correlated with each other. CTX and cell swelling elicit a rise of [Ca²⁺]_i whereas 1-EBIO and cell shrinkage induce a slight decrease of [Ca²⁺]_i in most MDCK-F cells. Taken together IK-channel-dependent perturbations of cell volume and anisotonicity elicit virtually identical effects on migration, actin filaments and [Ca²⁺]_i. We therefore suggest that cell volume – possibly via [Ca²⁺]_i– is the link between IK channel activity, actin filaments and migration. We propose a model for how temporal and local changes of cell volume can support the migration of MDCK-F cells. Received: 28 January 1999 / Received after revision: 15 April 1999 / Accepted: 16 April 1999     

Intracellular Ca2+ distribution in migrating transformed renal epithelial cells

 Migration of transformed Madin-Darby canine kidney (MDCK-F) cells depends on the polarized activity of a Ca²⁺-sensitive K⁺ channel. We tested whether a gradient of intracellular Ca²⁺ concentration ([Ca²⁺]_i) underlies the horizontal polarization of K⁺ channel activity. [Ca²⁺]_i was measured with the fluorescent dye fura-2/AM. Spatial analysis of [Ca²⁺]_i indicated that a horizontal gradient exists, with [Ca²⁺]_i being higher in the cell body than in the lamellipodium. Resting and maximal levels during oscillations of [Ca²⁺]_i in the cell body were found to be 135 ± 34 and 405 ± 59 nmol/l, respectively, whereas they were 79 ± 18 and 307 ± 102 nmol/l in the lamellipodium. This gradient can partially explain the preferential activation of K⁺ channels in the plasma membrane of the cell body. We applied a local superfusion technique during migration experiments and measurements of [Ca²⁺]_i to test whether its maintenance is due to an uneven distribution of Ca²⁺ influx into migrating MDCK-F cells. Locally superfusing the cell body of migrating MDCK-F cells with La³⁺ alone or together with charybdotoxin, a specific blocker of Ca²⁺-sensitive K⁺ channels, slowed migration to 47 ± 10% and 9 ± 5% of control, respectively. Local blockade of Ca²⁺ influx into the cell body and the lamellipodium with La³⁺ was followed by a decrease of [Ca²⁺]_i at both cell poles. This points to Ca²⁺ influx occurring over the entire cell surface. This conclusion was confirmed by locally superfusing Mn²⁺ over the cell body and the lamellipodium. Fura-2 fluorescence was quenched in both areas, the decrease of fluorescence being two to three times faster in the cell body than in the lamellipodium. However, this difference is insufficient to account for the observed gradient of [Ca²⁺]_i. We hypothesize that the polarized distribution of intracellular Ca²⁺ stores contributes significantly to the generation of a gradient of [Ca²⁺]_i. Received: 22 July 1996 / Received after revision: 17 December 1996 / Accepted: 10 January 1997     

A transient,RCK4-like K+ current in cultured Xenopus olfactory bulb neurons

 A transient K⁺ current in cultured olfactory bulb neurons of Xenopus tadpoles was studied using the whole-cell patch-clamp technique. The current, which was resistant to 80 mM tetraethylammoniumchloride (TEA) and 10 nM charybdotoxin but blocked by 5 mM 4-aminopyridine (4-AP), activated between −60 and −40 mV and showed time- and voltage-dependent inactivation. Its peak amplitude was nearly independent of the extracellular K⁺ concentration ([K⁺]_o) in the range of 0.05 to 10 mM, indicating that its conductance increased upon increasing [K⁺]_o. The transient K⁺ current showed a slow recovery from inactivation with the time for half-maximum recovery from a conditioning pulse to 80 mV for 1 s varying from 100 ms to 500 ms. Complete recovery required as much as 5–10 s at −80 mV, but could be speeded up at hyperpolarized potentials. The current resembles the RCK4 (Kv1.4) current of rat neurons except that its recovery from inactivation was independent of [K⁺]_o. High-freqency stimulation (20–67 Hz) of the neurons with short (5 ms) voltage pulses resulted in a frequency-dependent, progressive inactivation of the transient K⁺ current. This suggests that, during phasic responses of olfactory bulb neurons, inactivation of the transient K⁺ current occurs and may lead to lengthening of action potentials and facilitation of synaptic transmission. Received: 21 January 1996 / Received after revision: 22 May 1996 / Accepted: 7 June 1996     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

Polarized ion transport during migration of transformed Madin-Darby canine kidney cells

Epithelial cells lose their usual polarization during carcinogenesis. Although most malignant tumours are of epithelial origin little is known about ion channels in carcinoma cells. Previously, we observed that migration of transformed Madin-Darby canine kidney (MDCK-F) cells depended on oscillating K⁺ channel activity. In the present study we examined whether periodic K⁺ channel activity may cause changes of cell volume, and whether K⁺ channel activity is distributed in a uniform way in MDCK-F cells. After determining the average volume of MDCK-F cells (2013±270 m³; n=8) by means of atomic force microscopy we deduced volume changes by calculating the K⁺ efflux during bursts of K⁺ channel activity. Therefore, we measured the membrane conductance of MDCK-F cells which periodically rose by 22.3±2.5 nS from a resting level of 6.5±1.4 nS (n=12), and we measured the membrane potential which hyperpolarized in parallel from –35.4±1.2 mV to –71.6±1.8 mV (n=11). The distribution of K⁺ channel activity was assessed by locally superfusing the front or rear end of migrating MDCK-F cells with the K⁺ channel blocker charybdotoxin (CTX). Only exposure of the rear end to CTX inhibited migration providing evidence for horizontal polarization of K⁺ channel activity in transformed MDCK-F cells. This is in contrast to the vertical polarization in parent MDCK cells. We propose that the asymmetrical distribution of K⁺ channel activity is a prerequisite for migration of MDCK-F cells.     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

Characterization of an inward-rectifying potassium current in NG108-15 neuroblastoma×glioma cells

 By using the whole-cell patch-clamp technique, an inwardly rectifying potassium current, which resembled the ”classic” inward-rectifying potassium current (I _KIR) of other cells in terms of electrophysiological and pharmacological properties, was identified in db-cAMP-differentiated NG108-15 cells. First, the current was dependent on voltage and time. It could be elicited by applying an initial depolarizing prepulse and a subsequent hyperpolarizing command pulse to the cell. The amplitude of the current depended on both the prepulse and the command pulse and increased with the hyperpolarization of the command pulse as well as the depolarization and the prolongation of the prepulse. The activation and inactivation of the current could be fitted well by single-exponential functions and increased with the hyperpolarization of the membrane. Second, the current was dependent on the extracellular potassium concentration ([K⁺]_o). Elevation of [K⁺]_o resulted in a marked increase in the current amplitude and a positive shift of the peak-current/voltage curve as well as the reversal potential. A tenfold increase of [K⁺]_o introduced an ≈43-mV shift of the reversal potential, indicating that the current was carried mainly by K⁺. The conductance (g/g _Max) of the current was also dependent on the [K⁺]_o and increased with increases in [K⁺]_o in a manner approximately proportional to the square-root of [K⁺]_o. Finally, the current was sensitive to Cs⁺ (1 mmol/l), Ba²⁺ (1 mmol/l) and quinidine (0.2 mmol/l); whereas, two typical potassium channel inhibitors, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), were weak blockers and reduced the current at high concentration (>10 mmol/l). It was also observed that the current was depressed by Cd²⁺ (1 mmol/l) and Co²⁺ (1 mmol/l) and increased by perfusing the cell with Ca²⁺-free solution. Thus, except for the sensitivity to Cd²⁺, Co²⁺ and Ca²⁺, the current displayed most of the hallmarks described for the ”classic”I _KIR. In conclusion, there appears to be a voltage-dependent I _KIR-type inward rectifier in db-cAMP-differentiated NG108-15 cells. Received: 3 June 1996 / Received after revision: 31 October 1996 / Accepted: 1 November 1996     

The sustained inward current in sino-atrial node cells of guinea-pig heart

 Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current I _f , the delayed rectifier K⁺ current I _K and the L-type Ca²⁺ current I _Ca,L as well as by the absence of both the transient outward current I _to and the inward rectifier K⁺ current I _K,1. After blocking I _f and I _K, depolarizing pulses from –80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca²⁺] ([Ca²⁺]_o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na⁺]_o, differently from I _Ca,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of I _st described in the rabbit SA node pacemaker cells, and that I _st is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither I _f nor I _st. Received: 22 July 1996 / Received after revision: 30 September 1996 / Accepted: 9 October 1996     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

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.     

Characterization of whole-cell currents in mucosal and connective tissue rat mast cells using amphotericin-B-perforated patches and temperature control

 Rat mucosal type mast cells are thought to possess only a K⁺-selective inwardly rectifying (IR_K) current in the resting state. We used rat-bone-marrow-derived mast cells (BMMCs) as a model of mucosal mast cells and recorded whole-cell membrane currents from cells perforated with amphotericin B. Under these conditions, both inwardly rectifying (IR) and outwardly rectifying (OR) currents were observed. The reversal potential and conductance of the IR current depended on the extracellular K⁺ concentration, indicating that the channel was K⁺ selective. The OR current was not affected by changes in extracellular K⁺ concentration, but lowering extracellular Cl^–concentration reduced the conductance and shifted the reversal potential in a positive direction. The OR current was not affected by K⁺ channel blockers, but was reversibly blocked by the chloride channel blocker 4,4’-diisothiocyanato-2,2’-stilbenedisulphonate (DIDS), again indicating a Cl^–conductance. The IR_K current was also detected in the majority of cells using the conventional whole-cell recording configuration at room temperature. In contrast, the OR_Cl current was only observed in 7% of recordings made at room temperature with the conventional whole-cell voltage-clamp mode, but was detected in 66% of cells if the bath temperature was increased and the integrity of the cell’s cytoplasm was preserved by using the perforated-patch technique. Under similar conditions, the OR_Cl current was also present in rat peritoneal mast cells, a connective tissue phenotype previously thought to have no whole-cell currents in the resting state. The role of this current and factors affecting its activation are discussed. Received: 10 May 1996 / Received after revision: 4 July 1996 / Accepted: 8 July 1996     

Roles of the voltage-gated K+ channel subunits, Kv 1.5 and Kv 1.4, in the developmental changes of K+ currents in cultured neonatal rat ventricular cells

 To investigate the roles of voltage-gated K⁺ channel subunits, Kv 1.5 and Kv 1.4, in the developmental regulation of K⁺ currents, we determined the K⁺ channel activities and the distributions of K⁺ channel subunits in the same single cultured neonatal rat ventricular cells, using a whole-cell patch-clamp technique and an immunocytochemical analysis of K⁺ channel proteins. In 5-day cultured cells, two types of 4-aminopyridine (4-AP)-sensitive and rapidly activating K⁺ currents, the transient outward current (I_to) and the ultrarapid delayed rectifier (I_Kur), could be distinguished. A small proportion of 5-day cells expressing sole I_Kur demonstrated an intense anti-Kv 1.5 antibody labeling with punctate distribution outlining the cells, while a weak staining was observed in the majority of 5-day cells expressing sole I_to. At day 15 of cell culture, only I_to was present with a lower level of the immunocytochemical expression of Kv 1.5 channel protein. Staining of the Kv 1.4 channel protein was qualitatively similar in the 5-day cells expressing either I_to or I_Kur. However, anti-Kv 1.4 antibody did not label the 15-day cultured cells showing remarkably increased I_to density. Our results strongly indicate that the Kv 1.5 channel expression may underlie the developmental regulation of I_Kur, while Kv 1.4 channel does not contribute to the postnatal increase in I_to. Received: 19 December 1996 / Received after revision: 9 February 1997 / Accepted: 19 February 1997     

Inwardly rectifying K+ currents of alveolar type II cells isolated from fetal guinea-pig lung: regulation by G protein- and Mg2+-dependent pathways

 K⁺ currents in alveolar type II cells, isolated from fetal guinea-pig lung, were studied using the whole-cell patch-clamp technique. Inwardly rectifying (IR) K⁺ currents were observed when cells were bathed in symmetrical KCl-rich solutions. When extracellular K⁺ was replaced by Na⁺, inward currents were greatly decreased and the zero-current potential moved from 0 mV to –69 mV, indicating high K⁺ selectivity. In recordings with an intracellular KCl-rich solution, containing 1.12 mM Mg²⁺ and 10^–8 M free Ca²⁺, IR K⁺ currents slowly diminished with time. Addition of the irreversible G protein activator, guanosine 5’-O-(3-thiotriphosphate) (GTP [γ-S]), to the intracellular solution accelerated the rate of current run-down. In experiments where the intracellular solution was Mg²⁺ free, current run-down was abolished. The rate of current run-down was found to increase with increasing free intracellular [Mg²⁺]. Raising the intracellular free [Ca²⁺] to 10^–6 M under Mg²⁺-free conditions had no effect on the K⁺ currents. Extracellular Ba²⁺ blocked the IR K⁺ currents in a concentration- and voltage-dependent manner. Tolbutamide, a blocker of ATP-sensitive K⁺ (K_ATP) channels, had no effect on the currents. The single channel underlying the whole-cell IR K⁺ currents displayed inward rectification and had a conductance of 31 pS in symmetrical KCl-rich solutions. We demonstate that mRNA coding for IRK1 is expressed in this cell preparation. Possible functions for this channel are discussed. Received: 28 June 1996 / Received after revision and accepted: 9 September 1996     

Effect of verapamil on restoration of cardiac performance in raised [K+]0 by adrenergic stimulation in the rabbit

Modulation of the L-type calcium channel by catecholamines improves action potential parameters in single ventricular myocytes depolarized by high [K⁺]₀ Tyrode. Whether this modulation is important in offsetting the negative effects of hyperkalaemia in the whole heart is not known. We tested the effects of the calcium channel antagonist, verapamil, on restoration of cardiac performance by adrenergic stimulation in high [K⁺]₀ in anaesthetized rabbits and isolated perfused working rabbit hearts. Raised [K⁺]₀ decreased SBP, LVP and LVdP/dt_maxin vivo ([K⁺]_a 8.6 ± 0.2 mM; n= 10) and aortic flow (AF) in the isolated heart (8 mM [K⁺]₀ Tyrode; n= 25). However, the negative effects of raised [K⁺]_a were offset by isoprenaline (Iso, 1 μg kg^-1 min^-1 i.v.) in vivo and by noradrenaline (NA, 80 nM) in the isolated heart. Verapamil (0.15 mg kg^-1 iv.; 15 nM isolated heart) markedly potentiated the negative inotropic effects of raised [K⁺]_n in both preparations. Verapamil attenuated the effect of isoprenaline in vivo but in the isolated heart, the protective effect of NA in 8 mM [K⁺] Tyrode (AF 97 ± 10 mL min¹ in 8 mM [K⁺]₀ compared with AF 141 ± 8.5 mL min^-1 in 8 mM [K⁺]₀+ NA) was offset by the drug (90±8mL min^-1 in 8 mM [K⁺]₀+ NA + V). Furthermore, verapamil abolished aortic flow in 8 mM [K⁺]₀ alone. These findings suggest that the heart may be critically dependent on modulation of intracellular calcium in order to tolerate concentrations of K⁴ similar to those seen during a short burst of intensive exercise ([K⁺]_a 8.6 mM).     

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     

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na⁺–K⁺ pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na⁺]_i, [K⁺]_i, and [Cl⁻]_i, as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na⁺]_i increase, a [K⁺]_i decrease, and a membrane depolarization, and it also evoked an increase in [Cl⁻]_i. The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na⁺ or Cl⁻, NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na⁺–K⁺ pump was inhibited by omitting bath K⁺. Also, in K⁺-free solution, [Na⁺]_i increased and [K⁺]_i dropped, but [Cl⁻]_i slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl⁻ gradient.     

Modulation of large conductance Ca2+-activated K+ channel by Gαh (transglutaminase II) in the vascular smooth muscle cell

 Among G-proteins, G_h is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca²⁺-activated K⁺ (Maxi-K⁺) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPγS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPβS resulted in reversal of the GTPγS effect. When the Gα_h7 antibody was applied, the GTPγS-stimulated channel activity was significantly inhibited to control level, suggesting that Gα_h is involved in activation of the Maxi-K⁺ channel in smooth muscle cells. Received: 23 September 1996 / Received after revision: 26 November 1996 / Accepted: 3 December 1996     

Monovalent cation conductance of the sustained inward current in rabbit sinoatrial node cells

 Under the whole cell clamp, superfusion of the rabbit sinoatrial node cells with a Na⁺-free solution suppressed the sustained inward current (I_st), and the L-type Ca²⁺ current (I_Ca,L) could be recorded on depolarization less negative than –40 mV from the holding potential of –80 mV. On the other hand, replacement of Ca²⁺ with Mg²⁺ in the external solution suppressed inward-going I_Ca,L and isolated I_st. Under this condition, I_st measured as a nicardipine-sensitive current showed an activation threshold between –60 and –70 mV. The conductance sequence of I_st for monovalent ions was determined as Na⁺ > Li⁺ >> K⁺ @ Cs⁺ by replacing the external Na⁺ with these alkali metal ions. The contribution of I_st to the diastolic depolarization is discussed. Received: 12 June 1996 / Received after revision: 31 July 1996 / Accepted: 7 August 1996     

Neomycin inhibits K+-induced force and Ca2+ channel current in rat arterial smooth muscle

 The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K⁺-induced isometric force and voltage-gated Ca²⁺ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca²⁺ channel antagonist (–)202–791 concentration-dependently and reversibly inhibited 40 mM K⁺-induced isometric force in rings of rat mesenteric and basilar arteries (IC₅₀ values of 70 μM and 1.2 nM, respectively, n = 10 and 4). Elevation of [Ca²⁺]_o by a factor of 2 significantly reduced the IC₅₀ values for inhibition of K⁺-induced force for both neomycin and (–)202–791 (192 μM and 3.7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba²⁺ was reversibly and concentration-dependently inhibited by neomycin (IC₅₀ 32 μM, n = 3). The concentration/effect curve for inhibition of the inward Ba²⁺ current by neomycin was significantly shifted to the right when [Ba²⁺]_o was raised from 1.8 mM to 10 mM (IC₅₀ 144 μM, n = 8). Our findings suggest that neomycin relaxes high-K⁺-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca²⁺ channels. Received: 1 August 1996 / Received after revision and accepted: 11 September 1996     

A calcium-dependent chloride current in insulin-secreting βTC-3 cells

 Ca²⁺-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K⁺ channel has received the most attention, while a low-conductance Ca²⁺-activated K⁺ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca²⁺-dependent Cl^–current [I _Cl(Ca)] in insulin-secreting pancreatic β cells. The reported I_Cl(Ca) could be activated under physiological Ca²⁺ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca²⁺ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca²⁺ current (I _Ca) present in these cells; they were blocked completely by the removal of external Ca²⁺ or application of Cd²⁺ at concentrations sufficient for complete block of I _Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of I _Ca showed a weak linear voltage dependence, while at voltages positive to activation of I _Ca it followed the voltage dependence of I _Ca. This Ca²⁺-dependent current reversed at –21.5 mV and when the external Cl^–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl^–-selective current. Cl^–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca²⁺-dependent Cl^–current [I _Cl(Ca)]. From substitution of the external Cl^–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I _Cl(Ca): I ^–>NO₃ ^–>Br^–>Cl^–>Acetate^–. Received: 10 October 1996 / Received after revision and accepted: 19 December 1996