Differential segmental activation of Ca-dependent CIand Kchannels in pulmonary arterial myocytes

Differential segmental distribution of electrophysiologically distinct myocytes helps to explain the variability of the pulmonary arteries to vasoactive agents. We have studied whether Ca²⁺-dependent CI⁻(CI_Ca) and K⁺(K_Ca) channels are activated differentially in enzymatically dispersed conduit and resistance myocytes. We measured cytosolic [Ca²⁺] and the changes of membrane current and potential elicited by spontaneous or agonist-induced Ca²⁺oscillations. Conduit arteries contained a heterogeneous cell population with a variable mixture of K_Caand CI_Caconductances. Resistance arteries contained a more homogeneous cell population with predominance of CI_Cachannel activation. The relation between K_Caand CI_Caconductances in a given conduit myocyte determines the size of the V_mchange in response to a rise of cytosolic [Ca²⁺]. Conduit myocytes tend to hyperpolarize towards the K⁺equilibrium potential ( − 90 m V). In resistance myocytes, release of Ca²⁺from stores activates CI_Cachannels and brings V_mto a value close to the chloride equilibrium potential ( − 20 or − 30 m V) thus favouring opening of Ca²⁺channels and Ca²⁺influx. In resistance vessels CI_Cachannels contribute to link agonist-induced Ca²⁺release from stores and membrane depolarization, thus permitting protracted vasoconstriction.     

Large conductance, Ca-activated K channels (BKCa) and arteriolar myogenic signaling

Myogenic, or pressure-induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this vascular smooth muscle (VSM) response are events including membrane depolarization, Ca²⁺ entry and mobilization, and activation of contractile proteins. Large conductance, Ca²⁺-activated K⁺ channel (BK_Ca) has been implicated in several of these steps including, (1) channel closure causing membrane depolarization, and (2) channel opening causing hyperpolarization to oppose excessive pressure-induced vasoconstriction. As multiple mechanisms regulate BK_Ca activity (subunit composition, membrane potential (Em) and Ca²⁺ levels, post-translational modification) tissue level diversity is predicted. Importantly, heterogeneity in BK_Ca channel activity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BK_Ca channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation.     

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK_Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK_Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK_Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca²⁺ and voltage without altering the unitary conductance and the K⁺ selectivity. Our results suggest a novel mechanism by which exercise training increases the K⁺ currents by changing the BK_Ca channel activities and kinetics.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

Summary A barium-sensitive Ca-activated K⁺ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K⁺ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K⁺ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K⁺-channel activity is assayed by measurement of⁸⁶Rb⁺ uptake against a large opposing K⁺ gradient. The reconstituted K⁺ channel is activated by Ca²⁺ in the physiological range of concentration (K_1/22×10^–7 m at pH 7.2) as found for the K⁺ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba²⁺, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K⁺ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K⁺-channel protein.Titration with Ca²⁺ shows that most of the active K⁺ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca²⁺ ionophore in order to observe Ca²⁺ stimulation. The reconstituted K⁺ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K⁺ channel in absence of Ca²⁺, to the level of activity seen with saturating concentrations of Ca²⁺. This tryptic split is located in a cytoplasmic aspect of the K⁺ channel that appears to be involved in opening and closing the K⁺ channel in response to Ca²⁺ binding.     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

Transient receptor potential vanilloid 4 (TRPV4) channels are Ca²⁺-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca²⁺ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca²⁺ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca²⁺ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type Ca_V1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive G_q-coupled receptor (opto-α₁AR) resulted in TRPV4-mediated Ca²⁺ influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca²⁺ influx into arterial myocytes.     

Sarcoplasmic Reticulum K (TRIC) Channel Does Not Carry Essential Countercurrent during Ca Release

The charge translocation associated with sarcoplasmic reticulum (SR) Ca²⁺ efflux is compensated for by a simultaneous SR K⁺ influx. This influx is essential because, with no countercurrent, the SR membrane potential (V_m) would quickly (<1 ms) reach the Ca²⁺ equilibrium potential and SR Ca²⁺ release would cease. The SR K⁺ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K⁺ countercurrent during release. To better define the physiological role of the SR K⁺ channel, we compared SR Ca²⁺ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K⁺ channel function. Specifically, we reduced SR K⁺ channel conduction 35 and 88% by replacing cytosolic K⁺ for Na⁺ or Cs⁺ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca²⁺ reloading, and caffeine-evoked Ca²⁺ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K⁺ (TRIC) channels is not required to support SR Ca²⁺ release (or uptake). Because K⁺ enters the SR through RyRs during release, the SR K⁺ (TRIC) channel most likely is needed to restore trans-SR K⁺ balance after RyRs close, assuring SR V_m stays near 0 mV.     

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch–clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca²⁺-activated K⁺-selective channel K_Ca3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca²⁺] will cause mtK_Ca3.1 opening, thus linking inner membrane K⁺ permeability and transmembrane potential to Ca²⁺ signalling.     

Sarcoplasmic Reticulum K+ (TRIC) Channel Does Not Carry Essential Countercurrent during Ca2+ Release

The charge translocation associated with sarcoplasmic reticulum (SR) Ca²⁺ efflux is compensated for by a simultaneous SR K⁺ influx. This influx is essential because, with no countercurrent, the SR membrane potential (V_m) would quickly (<1 ms) reach the Ca²⁺ equilibrium potential and SR Ca²⁺ release would cease. The SR K⁺ trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K⁺ countercurrent during release. To better define the physiological role of the SR K⁺ channel, we compared SR Ca²⁺ transport in saponin-permeabilized cardiomyocytes before and after limiting SR K⁺ channel function. Specifically, we reduced SR K⁺ channel conduction 35 and 88% by replacing cytosolic K⁺ for Na⁺ or Cs⁺ (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca²⁺ reloading, and caffeine-evoked Ca²⁺ release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K⁺ (TRIC) channels is not required to support SR Ca²⁺ release (or uptake). Because K⁺ enters the SR through RyRs during release, the SR K⁺ (TRIC) channel most likely is needed to restore trans-SR K⁺ balance after RyRs close, assuring SR V_m stays near 0 mV.     

Emerging Role of the Calcium-Activated,Small Conductance,SK3 K+ Channel in Distal Tubule Function: Regulation by TRPV4

The Ca²⁺-activated, maxi-K (BK) K⁺ channel, with low Ca²⁺-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K⁺ secretion. In the present study we demonstrate that the Ca²⁺-activated, SK3 (K_Ca2.3) K⁺ channel, with high Ca²⁺-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K⁺ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca²⁺-dependent K⁺ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca²⁺-permeable TRPV4 channel, thereby inducing Ca²⁺ influx and elevating intracellular Ca²⁺ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca²⁺ influx, leading to elevated intracellular Ca²⁺ levels, activates this high Ca²⁺-affinity K⁺ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca²⁺-dependent regulation of membrane potential and K⁺ secretion.     

The effect of inactivation of calcium channels by intracellular Ca2+ ions in the bursting pancreatic β-cells

Based on recently determined ionic channel properties, a simple theoretical model for the burst activity of the pancreatic β-cell is formulated in this paper. The model contains an inward voltage-activated Ca²⁺ current which is inactivated by intracellular calcium ions and an outward K⁺ current that is activated by the membrane potential. The probability of opening of the channel gates is represented by Boltzmann equations. Our model is applicable in a regime where an ATP-blockable K⁺ channel is inhibited. In this regime, glucose is treated as an activator for the rate of efflux of intracellular Ca²⁺ ions, and hence its effect is equated tok _Ca, the efflux rate constant. In addition, intracellular H⁺ ion, which is a byproduct of the glycolytic metabolic process, is treated as a competitive inhibitor for Ca²⁺ ion. Since H⁺ is a competitive inhibitor (according to our assumption), its effect is equated to the strength of the Ca_i dissociation constantK _h. In the model, a Ca²⁺ binding site is assumed to exist in the inner membrane of the voltage-gated Ca²⁺ channel. The model predicts that a spike and burst electrical pattern can be generated by varyingk _ca and that a given pattern may produce different levels of intracellular Ca²⁺ depending onK _h. In other words, it predicts that levels of [Ca²⁺]_i can be separated from the electrical activity by controlling the concentration of glucose and pH appropriately. This may account for the experimental observation of Lebrun et al. (1985) that insulin secretion is not correlated to the burst of electrical activity.     

Light-induced cytosolic calcium transients in green plant cells. ll. The effect on a K+ channel as studied by a kinetic analysis in Chara corallina

The fluorescent dye chlorotetracycline was used to study the relationship between the light-induced decrease in cytosolic free calcium concentration, [Ca²⁺]_c, and its effect on ion transport at the plasma membrane in the giant cells of Chara corallina Klein ex Willd. A kinetic analysis of the simultaneously measured light-induced changes in membrane potential and in [Ca²⁺]_c led to the same time constant of about 40 s. The reversal potential of the light effect on membrane potential was in agreement with the dominant role of a K⁺ channel in the plasma membrane. Thus, the experiments reported here provide evidence for the following light-driven signal transduction chain from the chloroplasts to K⁺ transport of the plasma membrane: (i) light causes an uptake of Ca²⁺ into the chloroplasts, (ii) this causes a decrease in cytosolic [Ca²⁺]_c, (iii) this leads to a decrease in the activity of a K⁺ channel. The results also initiated a re-analysis of previously published data of the light effect on the velocity of cytosolic streaming and supported the hypothesis that Ca²⁺ fluxes coming out of the chloroplasts upon darkening cause a Ca²⁺-induced phosphorylation of myosin, which slows down cytoplasmic streaming. Received: 3 May 1997 / Accepted: 19 May 1998     

The Xanthine Derivative KMUP-1 Attenuates Serotonin-Induced Vasoconstriction and K+-Channel Inhibitory Activity via the PKC Pathway in Pulmonary Arteries

Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K⁺ channel inhibition increases [Ca²⁺]_i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K⁺ channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K⁺ channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K⁺-channel (Kv1.5 and Kv2.1) and large-conductance Ca²⁺-activated K⁺-channel (BK_Ca) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K⁺-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH.     

Calcium-independent K+-selective channel from chromaffin granule membranes

Summary Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca²⁺ (1 mm). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mm K⁺), which was highly selective for K⁺ over Na⁺ (P _k/P _Na = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca²⁺] activated K⁺ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K⁺-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.     

Beauvericin-induced channels in ventricular myocytes and liposomes

The antibiotic Beauvericin (BEA) was previously shown to express ionophoric properties under simple experimental systems. Its channel-forming activity was examined in inside-out patches of ventricular myocytes and synthetic membranes with the patch clamp and fluorescence imaging techniques. Current transitions to several open state levels were evident after wash-in. The BEA channel is cation-selective. Conductance and kinetics are presented for K⁺ and Na⁺ substates and main states. The pore was blocked by La³⁺. In myocytes, the [K⁺]_i was reduced, while [Na⁺]_i and [Ca²⁺]_i increased, leading to cytolysis. These results indicate that BEA forms cation-selective channels in lipid membranes, which can affect the ionic homeostasis.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Dependence of membrane potential on Ca2+ transport in cultured cytotrophoblasts of human immature placentas

The electrical membrane properties of cultured human cytotrophoblast were examined by means of a standard electrophysiological technique. The mean values of the membrane potential (E_m) and the membrane resistance in a physiological medium were around ?49 mV and 12 MΩ, respectively. The membrane potential was dependent, to a large extent, on the external Ca²⁺ concentration ([Ca²⁺]₀). Deprivation of external Ca²⁺ reduced membrane potential to about ?20 mV, and an increase in [Ca²⁺]₀ caused a hyperpolarization in a saturable manner. The Ca²⁺-dependency of membrane potential was affected remarkably by [K⁺]₀, but not by [Na⁺]₀ or [Cl^?]₀. The intracellular Ca²⁺ injection hyperpolarized the membrane in a Ca²⁺-free medium. A Ca²⁺ channel blocker, verapamil, completely abolished the Ca²⁺-dependent E_m. The Ca²⁺-dependent E_m was also suppressed by cooling or by the application of metabolic inhibitors. It is suggested that the Ca²⁺-dependent E_m in cultured human cytotrophoblast is caused by a Ca²⁺ influx which, in turn, increases the K⁺ conductance of the cell membrane, presumably due to stimulation of Ca²⁺-activated K⁺ channel.