Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis

Plasma membrane potassium (K⁺) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying K⁺ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested the hypothesis that apoptotic proteins are linked with K⁺ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode revealed two components of voltage-gated outward K⁺ currents: one is sensitive to either tetraethylammonium (TEA) or tetrandrine (Tet), a maxi-conductance Ca²⁺-activated K⁺ (BK) channel blocker, and the other is sensitive to 4-aminopyridine (4-AP), a delayed rectifier K⁺ channel blocker. MTT and flow cytometry assays showed that TEA, Tet, or iberiotoxin (Ibtx), a selective BK channel blocker, inhibited HeLa and A2780 cell proliferation in a dose-dependent manner with G₁ phase arrest. Pretreatment with TEA or Tet also induced apoptosis in HeLa and A2780 cells. However, glibenclamide (Gli), an ATP-sensitive K⁺ channel blocker, did not influence K⁺ currents, proliferation or apoptosis. Western blot analyses showed that while pretreatment of TEA and Tet produced an increase in expressions of p53, p21, and Bax, pretreatment of these two agents led to a decrease in expressions of heat shock protein (hsp)90α, hsp90β, and hsp70. Our results indicate that the blockade of BK channels results in tumor cell apoptosis and cycle arrest at G₁ phase, and the transduction pathway underlying the anti-proliferative effects is linked to the increased expression of apoptotic protein p53 and the decreased expression of its chaperone proteins hsp.     

KCa and Ca channels: The complex thought

Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca^2 +-activated K⁺ channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca^2 + channels are divided into two main families, voltage gated/voltage dependent Ca^2 + channels and non-voltage gated/voltage independent Ca^2 + channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca^2 + channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca^2 +. Non-voltage gated Ca^2 + channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca^2 + channels while non-voltage gated Ca^2 + channels are activated by Ca^2 + depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca^2 + channel families also control constitutive Ca^2 + entries. For reducing the energy consumption and for the fine regulation of Ca^2 +, KCa and Ca^2 + channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa–Ca^2 + channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current?

As in other salivary glands, the secretory cells of the sheep parotid have a resting K⁺ conductance that is dominated by BK channels, which are activated by acetylcholine (ACh) and are blocked by tetraethylammonium (TEA). Nevertheless, perfusion studies indicate that TEA does not inhibit ACh-evoked fluid secretion or K⁺ efflux from intact sheep parotid glands. In the present study, we have used whole-cell patch clamp techniques to show that ACh activates K⁺ and Cl^– conductances in sheep parotid secretory cells by increasing intracellular free Ca²⁺, and we have compared the blocker sensitivity of the ACh-evoked whole-cell K⁺ current to the previously reported blocker sensitivity of the BK channels seen in these cells.The ACh-induced whole-cell K⁺ current was not blocked by TEA (10 mmol/l) or verapamil (100 mol/l), both of which block the resting K⁺ conductance and inhibit BK channels in these cells. Quinine (1 mmol/l) and quinidine (1 mmol/l), although only weak blockers of the resting K⁺ conductance, inhibited the ACh-evoked current at 0 mV (K⁺ current), by 68% and 78%, respectively. 4-Aminopyridine (10 mmol/l) partially inhibited the ACh-induced K⁺ current and caused it to fluctuate. It also caused the resting membrane currents to fluctuate, possibly by altering cytosolic free Ca²⁺. Ba²⁺ (100 mol/l), a blocker of the inwardly rectifying K⁺ conductance in sheep parotid cells, had no effect on the ACh-induced K⁺ current.We conclude that the ACh-induced K⁺ conductance in sheep parotid cells is pharmacologically distinct from both the outwardly rectifying (BK) K⁺ conductance and the inwardly rectifying K⁺ conductance seen in unstimulated cells. Given that in vitro perfusion and K⁺ efflux studies on other salivary glands in which BK channels dominate the resting conductance (e.g., the rat mandibular, rat parotid and mouse mandibular glands) have revealed an insensitivity to TEA, suggesting that BK channels do not carry the ACh-evoked K⁺ current, we propose that BK channels do not contribute substantially to the K⁺ current evoked by ACh in the secretory cells of most salivary glands.This project was supported by the Australian Research Council. We thank Dr. N. Sangster, Dr. J. Rothwell and Mr. R. Murphy for giving us access to their sheep.     

The role of hERG1 K channels and a functional link between hERG1 K channels and SDF-1 in acute leukemic cell migration

Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4, regulate stem/progenitor cell migration and retention in the bone marrow and are required for hematopoiesis. Recent studies found that hERG1 K⁺ channels were important regulators of tumor cell migration. In this study, we investigated whether SDF-1 induced acute leukemic cell migration associated with hERG1 K⁺ channels. Our results showed that E-4031, a specific hERG1 K⁺ channels inhibitor, significantly blocked SDF-1-induced migration of leukemic cell lines, primary acute leukemic cells, leukemic stem cells and HEK293T cells transfected with herg-pEGFP. The migration of phenotypically recognizable subsets gave the indication that lymphoblastic leukemic cells were inhibited more than myeloid cells while in the presence of E-4031 which maybe associated with herg expression. SDF-1 increased hERG1 K⁺ current expressed in oocytes and HEK293T cells transfected with herg-pEGFP. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless if untreated or treated with E-4031 for 24 h (P > 0.05). The hERG1 K⁺ current increased by SDF-1 might contribute to the mechanism of SDF-1-induced leukemic cell migration. The data suggested that hERG1 K⁺ channels functionally linked to cell migration induced by SDF-1.     

K+ Channel Expression in Human Breast Cancer Cells: Involvement in Cell Cycle Regulation and Carcinogenesis

K⁺ channels are a most diverse class of ion channels in the plasma membrane and are distributed widely throughout a variety of cells including cancer cells. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels and that these K⁺ channels play important roles in regulating tumor cell proliferation, cell cycle progression and apoptosis. Moreover, a significant increase in K⁺ channel expression has been correlated with tumorigenesis, suggesting the possibility of using these proteins as transformation markers and perhaps reducing the tumor growth rate by selectively inhibiting their functional activity. Significant progress has been made in defining the properties of breast K⁺ channels, including their biophysical and pharmacological properties and distribution throughout different phases of the cell cycle in breast cell line MCF-7. This review aims to provide a comprehensive overview of the current state of research into K⁺ channels/currents in breast cancer cells. The possible mechanisms by which K⁺ channels affect tumor cell proliferation and cell cycle progression are discussed.     

Ion channels in cancer: future perspectives and clinical potential

Ion transport across the cell membrane mediated by channels and carriers participate in the regulation of tumour cell survival, death and motility. Moreover, the altered regulation of channels and carriers is part of neoplastic transformation. Experimental modification of channel and transporter activity impacts tumour cell survival, proliferation, malignant progression, invasive behaviour or therapy resistance of tumour cells. A wide variety of distinct Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels have been implicated in tumour growth and metastasis. Further experimental information is, however, needed to define the specific role of individual channel isoforms critically important for malignancy. Compelling experimental evidence supports the assumption that the pharmacological inhibition of ion channels or their regulators may be attractive targets to counteract tumour growth, prevent metastasis and overcome therapy resistance of tumour cells. This short review discusses the role of Ca²⁺ permeable channels, K⁺ channels, Na⁺ channels and anion channels in tumour growth and metastasis and the therapeutic potential of respective inhibitors.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: Evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK_Ca channels opener NS1619 reversed thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; BK_Ca channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca²⁺ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK_Ca channels may play a regulatory role in it. (Mol Cell Biochem 269: 37–47, 2005)     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

The role of Eag and HERG channels in cell proliferation and apoptotic cell death in SK-OV-3 ovarian cancer cell line

Background  

The voltage gated potassium (K⁺) channels Eag and HERG have been implicated in the pathogenesis of various cancers, through association with cell cycle changes and programmed cell death. The role of these channels in the onset and progression of ovarian cancer is unknown. An understanding of mechanism by which Eag and HERG channels affect cell proliferation in ovarian cancer cells is required and therefore we investigated their role in cell proliferation and their effect on the cell cycle and apoptosis of ovarian cancer cells.     

Current Transients Associated with BK Channels in Human Glioma Cells

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, I _transient, activated and inactivated rapidly (1 ms). The I-V relationship of I _transient had features that were inconsistent with simple ionic current through open ion channels: (i) I _transient amplitude peaked with a –80 mV voltage change and was invariant over a 200 mV range, and (ii) I _transient remained large and outward at –140 mV. We provide evidence for a direct relationship of I _transient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and I _transient seen over time were often perfectly in phase. Substituting intracellular K⁺ with Cs⁺, Li⁺, or Na ⁺ ions reversibly reduced I _transient and BK currents. I _transient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting I _transient is unique to BK currents in human glioma cells. We conclude that I _transient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K⁺ ions are trapped within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.     

Satellite glial cells In Situ within mammalian prevertebral ganglia express K+ channels active at rest potential

Patch-clamp experiments were performed on satellite glial cells wrapped around sympathetic neurons in the rabbit coeliac ganglion. With the cleaning method used, the glial cells could be kept in place and were directly accessible to the patch-clamp pipettes. Whole-cell recordings showed that glial cells had almost ohmic properties. Their resting potential (–79.1±1.2 mV) was found to be very nearly the same as the K⁺ reversal potential and 20 mV more negative than that of the neurons they encapsulated. Unitary currents from ionic channels present in the glial membrane were recorded in the cell-attached configuration with pipettes filled with various amounts of K⁺, Na⁺ and gluconate. Only K⁺-selective channels with slight inwardly rectifying properties (in the presence of 150 mM [K⁺]₀) were detected. These channels were active (P ₀=0.7–0.8) at the cell resting potential. The channel conductance, but not its opening probability, was dependent on the [K⁺] in the pipette. Cl^–-selective channels (outwardly rectifying and large conductance channels) were detected in excised patches.The properties of the K⁺ channels (increased inward current with [K⁺] and detectable outward current at low [K⁺]) are well suited for siphoning the K⁺ released by active neurons.     

The potassium channel opener NS1619 modulates calcium homeostasis in muscle cells by inhibiting SERCA

NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel opener. It was previously reported that activation of BK_Ca channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca²⁺ homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca²⁺ homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca²⁺ uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca²⁺ from internal stores in a concentration-dependent manner. Ca²⁺ accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca²⁺ concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered.     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.     

Hypoxic preconditioning induces neuroprotection against transient global ischemia in adult rats via preserving the activity of Na(+)/K(+)-ATPase

We demonstrated previously that 30 min of hypoxic preconditioning (HPC) applied 1 day before 10 min of transient global cerebral ischemia (tGCI) reduced neuronal loss in the hippocampal CA1 subregion in adult rats. The aim of the present study was to investigate the role of Na⁺/K⁺-ATPase and protein kinase Mζ (PKMζ) in the protective effect of HPC against tGCI in adult rats. We found that the activity of Na⁺/K⁺-ATPase decreased in the hippocampal CA1 subregion after 10 min of tGCI. This effect was not seen after 30 min of HPC in adult rats. Corresponding to the changes in Na⁺/K⁺-ATPase activity, the surface expression of Na⁺/K⁺-ATPase α1 subunit increased after HPC. Furthermore, HPC dramatically reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the hippocampal CA1 subregion after tGCI. However, neither PKMζ nor phosphorylation of PKMζ was changed after tGCI or HPC. The results of the present study are consistent with the hypothesis that both enhanced recovery of Na⁺/K⁺-ATPase activity due to preserved the protein levels of Na⁺/K⁺-ATPase α1 subunit and reduced DNA fragmentation after tGCI contribute to the protection afforded by HPC. However, PKMζ activation does not appear to play a role in this neuroprotection.     

Hemolymph ion regulation and kinetic characteristics of the gill (Na⁺, K⁺)-ATPase in the hermit crab Clibanarius vittatus (Decapoda, Anomura) acclimated to high salinity

We examine hemolymph ion regulation and the kinetic properties of a gill microsomal (Na⁺, K⁺)-ATPase from the intertidal hermit crab, Clibanarius vittatus, acclimated to 45‰ salinity for 10 days. Hemolymph osmolality is hypo-regulated (1102.5 ± 22.1 mOsm kg⁻¹ H₂O) at 45‰ but elevated compared to fresh-caught crabs (801.0 ± 40.1 mOsm kg⁻¹ H₂O). Hemolymph [Na⁺] (323.0 ± 2.5 mmol L⁻¹) and [Mg²⁺] (34.6 ± 1.0 mmol L⁻¹) are hypo-regulated while [Ca²⁺] (22.5 ± 0.7 mmol L⁻¹) is hyper-regulated; [K⁺] is hyper-regulated in fresh-caught crabs (17.4 ± 0.5 mmol L⁻¹) but hypo-regulated (6.2 ± 0.7 mmol L⁻¹) at 45‰. Protein expression patterns are altered in the 45‰-acclimated crabs, although Western blot analyses reveal just a single immunoreactive band, suggesting a single (Na⁺, K⁺)-ATPase α-subunit isoform, distributed in different density membrane fractions. A high-affinity (Vm = 46.5 ± 3.5 U mg⁻¹; K_0.5 = 7.07 ± 0.01 μmol L⁻¹) and a low-affinity ATP binding site (Vm = 108.1 ± 2.5 U mg⁻¹; K_0.5 = 0.11 ± 0.3 mmol L⁻¹), both obeying cooperative kinetics, were disclosed. Modulation of (Na⁺, K⁺)-ATPase activity by Mg²⁺, K⁺ and NH₄⁺ also exhibits site-site interactions, but modulation by Na⁺ shows Michaelis-Menten kinetics. (Na⁺, K⁺)-ATPase activity is synergistically stimulated up to 45% by NH₄⁺ plus K⁺. Enzyme catalytic efficiency for variable [K⁺] and fixed [NH₄⁺] is 10-fold greater than for variable [NH₄⁺] and fixed [K⁺]. Ouabain inhibited ≈80% of total ATPase activity (K_I = 464.7 ± 23.2 μmol L⁻¹), suggesting that ATPases other than (Na⁺, K⁺)-ATPase are present. While (Na⁺, K⁺)-ATPase activities are similar in fresh-caught (around 142 nmol Pi min⁻¹ mg⁻¹) and 45‰-acclimated crabs (around 154 nmol Pi min⁻¹ mg⁻¹), ATP affinity decreases 110-fold and Na⁺ and K⁺ affinities increase 2-3-fold in 45‰-acclimated crabs.