Gating pore currents,a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na⁺ and Ca²⁺ channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K⁺ ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K⁺ channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K⁺ (K2P) channels also contribute to the steady outward leak of K⁺ ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K⁺ channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

The Life and Death of Breast Cancer Cells: Proposing a Role for the Effects of Phytoestrogens on Potassium Channels

Changes in the regulation of potassium channels are increasingly implicated in the altered activity of breast cancer cells. Increased or reduced expression of a number of K⁺ channels have been identified in numerous breast cancer cell lines and cancerous tissue biopsy samples, compared to normal tissue, and are associated with tumor formation and spread, enhanced levels of proliferation, and resistance to apoptotic stimuli. Through knockout or silencing of K⁺ channel genes, and use of specific or more broad pharmacologic K⁺ channel blockers, the growth of numerous cell lines, including breast cancer cells, has been modified. In this manner it has been proposed that in MCF7 breast cancer cells proliferation appears to be regulated by the activity of a number of K⁺ channels, including the Ca²⁺ activated K⁺ channels, and the voltage-gated K⁺ channels hEAG and K_v1.1. The effect of phytoestrogens on K⁺ channels has not been extensively studied but yields some interesting results. In a number of cell lines the phytoestrogen genistein inhibits K⁺ current through several channels including K_v1.3 and hERG. Where it has been used, structurally similar daidzein has little or no effect on K⁺ channel activity. Since many K⁺ channels have roles in proliferation and apoptosis in breast cancer cells, the impact of K⁺ channel regulation by phytoestrogens is of potentially great relevance.     

Ion Channels in Cell Proliferation and Apoptotic Cell Death

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl⁻ and K⁺ channels. Besides regulating cytosolic Cl⁻ activity, osmolyte flux and, thus, cell volume, most Cl⁻ channels allow HCO₃⁻ exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K⁺ exit through K⁺ channels may decrease intracellular K⁺ concentration, which in turn favors apoptotic cell death. K⁺ channel activity further maintains the cell membrane potential, a critical determinant of Ca²⁺ entry through Ca²⁺ channels. Cytosolic Ca²⁺ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca²⁺ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.     

The chemiosmotic model of stomatal opening revisited

Stomata are light‐activated biological valves in the otherwise gas‐impermeable epidermis of aerial organs of higher plants. Stomata often regulate rates of photosynthesis and transpiration in ways that optimize whole‐plant carbon gain against water loss. Each stoma is flanked by a pair of opposing guard cells. Stomatal opening occurs by light‐activated increases in the turgor pressure of guard cells, which causes them to change shape so that the stomatal pore between them widens. These increases in turgor pressure oppose increases in cellular osmotic pressure that result from uptake of K⁺. K⁺ uptake occurs by a chemiosmotic mechanism in response to light‐activated extrusion of H⁺ outward across the plasma membrane of the guard cell. The initial changes in cellular membrane potential lead to the opening of inward‐rectifying K⁺ channels, after which K⁺ is taken up along its electrochemical gradient. Changes in membrane potential resulting from K⁺ uptake may be balanced by accumulation of Cl^?ions by guard cells and/or by synthesis of malic acid within each cell. Malic acid also acts to buffer increases in cytosolic pH caused by H⁺ extrusion. This review describes how the application of patch‐clamp technology to guard cell protoplasts has enabled investigators to elucidate the mechanisms by which H⁺ is extruded from guard cells, the types of ion channels present in the guard cell plasma membrane, how those ion channels are regulated, and the signal transduction processes that trigger stomatal opening and closing.     

Mitochondrial Ca2+-activated K+ channels and their role in cell life and death pathways

Ca²⁺-activated K⁺ channels (K_Ca) are expressed at the plasma membrane and in cellular organelles. Expression of all K_Ca channel subtypes (BK, IK and SK) has been detected at the inner mitochondrial membrane of several cell types. Primary functions of these mitochondrial K_Ca channels include the regulation of mitochondrial ROS production, maintenance of the mitochondrial membrane potential and preservation of mitochondrial calcium homeostasis. These channels are therefore thought to contribute to cellular protection against oxidative stress through mitochondrial mechanisms of preconditioning. In this review, we summarize the current knowledge on mitochondrial KCa channels, and their role in mitochondrial function in relation to cell death and survival pathways. More specifically, we systematically discuss studies on the role of these mitochondrial K_Ca channels in pharmacological preconditioning, and according protective effects on ischemic insults to the brain and the heart.     

Cell proliferation,potassium channels,polyamines and their interactions: a mini review

Polyamines, which are obligatory molecules involved in cell cycling and proliferation, are subject to a change in their free intracellular concentrations during the cell cycle. Potassium (K⁺) channels are also considered, but less well recognized, to be necessary for cell proliferation by either hyperpolarizing or depolarizing cells during the cell cycle. A block of polyamine synthesis as well as block or knockout of K⁺ channels can halt cell proliferation. K⁺ channels like BK (maxi calcium (Ca²⁺)-activated K⁺), Kir (inward rectifier), M-type K⁺-and TASK (two-pore domain K⁺) channels or the delayed rectifier K⁺ channels are modulated in their electrical properties by polyamines. Polyamines are most effective in blocking these channels when applied to the intracellular face of these channels except for TASK channels where they act only from the extracellular side. Quinidine, a general K⁺ channel blocker, was found to reduce putrescine concentrations, to block the ornithine decarboxylase and halt cell proliferation. From these results, the question arises if there is an interaction between polyamines, K⁺ channels and proliferation. It might be speculated that a decrease of intracellular polyamines allows more K⁺ channels to be active, thus inducing hyperpolarization, while an increase of the polyamine concentration may block K⁺ channel activity leading to depolarization of the membrane potential. On the other hand, a block or a deletion of K⁺ channels may cause a decrease of the polyamine concentration in cells. More research is needed to test these hypotheses.     

Potassium channel regulator KCNRG regulates surface expression of Shaker-type potassium channels

Besides their role in the generation of action potentials, voltage-gated potassium channels are implicated in cellular processes ranging from cell division to cell death. The K⁺ channel regulator protein (KCNRG), identified as a putative tumor suppressor, reduces K⁺ currents through human K⁺ channels hKv1.1 and hKv1.4 expressed in Xenopus oocytes. Current attenuation requires the presence of the N-terminal T1 Domain and immunoprecipitation experiments suggest association of KCNRG with the N-terminus of the channel. Our data indicates that KCNRG is an ER-associated protein, which we propose regulates Kv1 family channel proteins by retaining a fraction of channels in endomembranes.     

Pathophysiology of mitochondrial volume homeostasis: Potassium transport and permeability transition

Regulation of mitochondrial volume is a key issue in cellular pathophysiology. Mitochondrial volume and shape changes can occur following regulated fission-fusion events, which are modulated by a complex network of cytosolic and mitochondrial proteins; and through regulation of ion transport across the inner membrane. In this review we will cover mitochondrial volume homeostasis that depends on (i) monovalent cation transport across the inner membrane, a regulated process that couples electrophoretic K⁺ influx on K⁺ channels to K⁺ extrusion through the K⁺-H⁺ exchanger; (ii) the permeability transition, a loss of inner membrane permeability that may be instrumental in triggering cell death. Specific emphasis will be placed on molecular advances on the nature of the transport protein(s) involved, and/or on diseases that depend on mitochondrial volume dysregulation.     

Voltage-Gated K+ Channel,Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

Voltage-gated K⁺ (K_v) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between K_v channels and cell differentiation remains poorly investigated. This study demonstrates that K_v3.3 is involved in K562 cell erythroid differentiation. Down-regulation of K_v3.3 using siRNA-K_v3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of K_v3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The K_v channels, or at least K_v3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of K_v channel regulation of cell differentiation would provide important information regarding vital cellular processes.     

Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

ATP4A gene regulatory network for fine-tuning of proton pump and ion channels

The ATP4A encodes α subunit of H⁺, K⁺-ATPase that contains catalytic sites of the enzyme forming pores through cell membrane which allows the ion transport. H⁺, K⁺-ATPase is a membrane bound P-type ATPase enzyme which is found on the surface of parietal cells and uses the energy derived from each cycle of ATP hydrolysis that can help in exchanging ions (H⁺, K⁺ and Cl^?) across the cell membrane secreting acid into the gastric lumen. The 3-D model of α-subunit of H⁺, K⁺-ATPase was generated by homology modeling. It was evaluated and validated on the basis of free energies and amino acid residues. The inhibitor binding amino acid active pockets were identified in the 3-D model by molecular docking. The two drugs Omeprazole and Rabeprazole were found more potent interactions with generated model of α-subunit of H⁺, K⁺-ATPase on the basis of their affinity between drug–protein interactions. We have generated ATP4A gene regulatory networks for interactions with other proteins which involved in regulation that can help in fine-tuning of proton pump and ion channels. These findings provide a new dimension for discovery and development of proton pump inhibitors and gene regulation of the ATPase. It can be helpful in better understanding of human physiology and also using synthetic biology strategy for reprogramming of parietal cells for control of gastric ulcers.     

Direct visualization of KirBac3.1 potassium channel gating by atomic force microscopy

KirBac3.1 belongs to a family of transmembrane potassium (K⁺) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP₂), G-proteins, nucleotides (such as ATP) and ions (e.g. H⁺, Mg²⁺, Ca²⁺). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg²⁺, the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg²⁺, a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg²⁺, indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K⁺ channel. This is the first time AFM has been used as an interactive tool to study K⁺ channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.     

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.     

Expression of animal CED-9 anti-apoptotic gene in tobacco modifies plasma membrane ion fluxes in response to salinity and oxidative stress

Apoptosis, one form of programmed cell death (PCD), plays an important role in mediating plant adaptive responses to the environment. Recent studies suggest that expression of animal anti-apoptotic genes in transgenic plants may significantly improve a plant’s ability to tolerate a variety of biotic and abiotic stresses. The underlying cellular mechanisms of this process remain unexplored. In this study, we investigated specific ion flux “signatures” in Nicotiana benthamiana plants transiently expressing CED-9 anti-apoptotic gene and undergoing salt- and oxidative stresses. Using a range of electrophysiological techniques, we show that expression of CED-9 increased plant salt and oxidative stress tolerance by altering K⁺ and H⁺ flux patterns across the plasma membrane. Our data shows that PVX/CED-9 plants are capable of preventing stress-induced K⁺ efflux from mesophyll cells, so maintaining intracellular K⁺ homeostasis. We attribute these effects to the ability of CED-9 to control at least two types of K⁺-permeable channels; outward-rectifying depolarization-activating K⁺ channels (KOR) and non-selective cation channels (NSCC). A possible scenario linking CED-9 expression and ionic relations in plant cell is suggested. To the best of our knowledge, this study is the first to link “ion flux signatures” and mechanisms involved in regulation of PCD in plants.     

Properties of Shaker-type Potassium Channels in Higher Plants

Potassium (K⁺), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na⁺/K⁺ exchanger, which widely exists in animal cells, K⁺ channels and some type of K⁺ transporters function as K⁺ uptake systems in plants. Plant voltage-dependent K⁺ channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K⁺ channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K⁺ channels have been identified and play a crucial role in K⁺ homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K⁺ channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K⁺ channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K⁺ channels in plants in comparison to those of Shaker channels in animals and bacteria.     

Role of Voltage-Gated K+ (KV) Channels in Vascular Function

Voltage-dependent K⁺ (K_V) channels represent the most diverse group of K⁺ channels ubiquitously expressed in vascular smooth muscles. The K_V channels, together with other types of K⁺ conductances, such as Ca²⁺-activated (BK_Ca), ATP-sensitive (K_ATP), and inward rectifier, play an important role in the control of the cell membrane potential and regulation of the vascular contractility. Comparison of the expression of different K_V channel isoforms obtained from RT-PCR studies showed that virtually all K_V genes could be detected in vascular smooth muscle cells (VSMC). Based on the analysis of both mRNA and protein expressions, it is likely that K_V1.1, K_V1.2, K_V1.3, K_V1.5, K_V1.6, K_V2.1, and K_V3.1b channel isoforms are mainly responsible for the delayed rectifier current characterized electrophysiologically in most VSMC types studied to date. It has been recently demonstrated by our research group and by others that functional expression of multiple K_V channel α-subunits is not homogeneous and varies in different vascular beds of small and large arteries. Growing evidence suggests that in some small arteries, e.g., cerebral arteries and arterioles, the K_V channels are activated at more negative membrane voltages than BK_Ca, thus making a greater contribution to the control of vascular tone. Our data also suggest that in some blood vessels, such as the rat aorta and mouse small mesenteric arteries, the K_V channel current (identified mainly as passed through K_V2.1 channels), but not BK_Ca, is the predominant conductance activated even under conditions where intracellular Ca₂₊ concentration is increased up to 200 nM. In addition, our data indicate that the K_V2.1 channel current could also contribute to the regulation of the induced rhythmic activity in the rat aorta in vitro acting as a negative feedback mechanism for membrane depolarization. We and other experimenters also demonstrated that functional expression of K_V channels is a dynamic process, which is altered under normal physiological conditions (e.g., during the development of the vessels), and in various pathological states (e.g., pulmonary hypertension developing during chronic hypoxia). Recent findings also suggest that activation of K_V channels can also play a role in vascular apoptosis (causing loss of intracellular K⁺ and subsequent cell shrinking, one of the essential prerequisites of cellular apoptosis). To summarize, the K^V channels are essential for normal vascular function, and their expression and properties are altered under abnormal conditions. Therefore, understanding of the molecular identity of native K_V channels and their functional significance and elucidation of the mechanisms, which govern and control the expression of the K_V channels in the vasculature, represent an important and challenging task and could also lead to the development of useful therapeutic strategies for the treatment of cardiovascular diseases.     

Cell Volume Changes Regulate Slick (Slo2.1), but Not Slack (Slo2.2) K+ Channels

Slick (Slo2.1) and Slack (Slo2.2) channels belong to the family of high-conductance K⁺ channels and have been found widely distributed in the CNS. Both channels are activated by Na⁺ and Cl⁻ and, in addition, Slick channels are regulated by ATP. Therefore, the roles of these channels in regulation of cell excitability as well as ion transport processes, like regulation of cell volume, have been hypothesized. It is the aim of this work to evaluate the sensitivity of Slick and Slack channels to small, fast changes in cell volume and to explore mechanisms, which may explain this type of regulation. For this purpose Slick and Slack channels were co-expressed with aquaporin 1 in Xenopus laevis oocytes and cell volume changes of around 5% were induced by exposure to hypotonic or hypertonic media. Whole-cell currents were measured by two electrode voltage clamp. Our results show that Slick channels are dramatically stimulated (196% of control) by cell swelling and inhibited (57% of control) by a decrease in cell volume. In contrast, Slack channels are totally insensitive to similar cell volume changes. The mechanism underlining the strong volume sensitivity of Slick channels needs to be further explored, however we were able to show that it does not depend on an intact actin cytoskeleton, ATP release or vesicle fusion. In conclusion, Slick channels, in contrast to the similar Slack channels, are the only high-conductance K⁺ channels strongly sensitive to small changes in cell volume.     

NFκB mediated elevation of KCNJ11 promotes tumor progression of hepatocellular carcinoma through interaction of lactate dehydrogenase A

It has been well documented that changes in ion fluxes across cellular membranes is fundamental in maintaining cellular homeostasis. Dysregulation and/or malfunction of ion channels are critical events in the pathogenesis of diverse diseases, including cancers. In this study, we focused on the study of K⁺ channels in hepatocellular carcinoma (HCC). By data mining TCGA cohort, the expression of 27 K⁺ channels was investigated and KCNJ11 was identified as a key dysregulated K⁺ channels in HCC. KCNJ11 was differentially expressed in HCC and predicted a poor prognosis in HCC patients. Inhibition of NFκB signaling suppressed KCNJ11 expression in HCC cells. Knockdown of KCNJ11 expression inhibited cell proliferation, promoted cell apoptosis, and reduced cell invasive capacity. Mechanistically, we found that KCNJ11 promotes tumor progression through interaction with LDHA and enhancing its enzymatic activity. Pharmacological inhibition of LDHA largely compromised the oncogenic function of KCNJ11 in cell proliferation, cell apoptosis, and cell invasion. Collectively, our data, as a proof of principle, demonstrate that KCNJ11 acts as an oncogene in HCC though forming a complex with LDHA and suggest that targeting KCNJ11 can be developed as a candidate tool to dampen HCC.