[Show abstract][Hide abstract] ABSTRACT: Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.
[Show abstract][Hide abstract] ABSTRACT: The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca2+-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca2+ entry mediated by SACs presumably has a significant impact on various Ca2+-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca2+ influx via SACs triggered the activity of Ca2+-sensitive K+ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K+ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca2+ concentration due to SAC activity may provide a functional link between various Ca2+-dependent molecules in the processes of cellular mechanotransduction.
Biochemical and Biophysical Research Communications 08/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hydroethidine (HE) is a blue fluorescent dye which is intracellularly converted into red-emitting products upon two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells) and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells and causes cellular apoptosis in case of high HE concentrations and/or long cell incubations with HE, as well as high rate of HE oxidation in cells exposed to some stimuli.
Free Radical Biology and Medicine 07/2014; · 5.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A key role for podocytes in the pathogenesis of proteinuric renal diseases has been established. Angiotensin II causes depolarization and increased intracellular calcium concentration in podocytes; members of the cation TRPC channels family, particularly TRPC6, are proposed as proteins responsible for calcium flux. Angiotensin II evokes calcium transient through TRPC channels and mutations in the gene encoding the TRPC6 channel result in the development of focal segmental glomerulosclerosis. Here we examined the effects of angiotensin II on intracellular calcium ion levels and endogenous channels in intact podocytes of freshly isolated decapsulated mouse glomeruli. An ion channel with distinct TRPC6 properties was identified in wild-type, but was absent in TRPC6 knockout mice. Single-channel electrophysiological analysis found that angiotensin II acutely activated native TRPC-like channels in both podocytes of freshly isolated glomeruli and TRPC6 channels transiently overexpressed in CHO cells; the effect was mediated by changes in the channel open probability. Angiotensin II evoked intracellular calcium transients in the wild-type podocytes, which was blunted in TRPC6 knockout glomeruli. Pan-TRPC inhibitors gadolinium and SKF 96365 reduced the response in wild-type glomerular epithelial cells, whereas the transient in TRPC6 knockout animals was not affected. Thus, angiotensin II-dependent activation of TRPC6 channels in podocytes may have a significant role in the development of kidney diseases.Kidney International advance online publication, 19 March 2014; doi:10.1038/ki.2014.71.
Kidney International 03/2014; · 8.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dynamic remodeling of the actin cytoskeleton plays an essential role in cell migration and various signaling processes in living cells. One of the critical factors that controls the nucleation of new actin filaments in eukaryotic cells is the actin-related protein 2/3 (Arp2/3) complex. Recently, two novel classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate F-actin have been discovered and described. The current study aims at investigating the effects of CK-0944666 (CK-666) and its analogs (CK-869 and inactive CK-689) on the reorganization of the actin microfilaments in the cortical collecting duct cell line, M-1. We show that treatment with CK-666 and CK869 results in the reorganization of F-actin and drastically affects cell motility rate. The concentrations of the compounds used in this study (100-200 μM) neither cause loss of cell viability nor influence cell shape or monolayer integrity; hence, the effects of described compounds were not due to structural side effects. Therefore, we conclude that the Arp2/3 complex plays an important role in cell motility and F-actin reorganization in M-1 cells. Furthermore, CK-666 and its analogs are useful tools for the investigation of the Arp2/3 complex.
Cell and Tissue Research 09/2013; · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium signaling and Ca2+-conducting channels participate in development of immune response, cell proliferation, growth, and differentiation of lymphocytes. In this paper, the calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) were studied in the plasma membrane of the T cell line Jurkat and normal human blood lymphocytes (HBLs). The channels were spontaneously activated after removal of Ca2+ and Mg2+ from the surrounding solution, and were inactivated in the presence of the effective blocker of TRPV5 and TRPV6, ruthenium red. The current-voltage characteristics of the channels demonstrated an inward rectification. The channel activity in Jurkat cells was significantly higher than in normal HBLs. The real-time RT-PCR analysis revealed a higher level of mRNA of the genes encoding channels TRPV5 and TRPV6 in the proliferating Jurkat T-cells as compared with normal lymphocytes. In general, the data have shown that TRPV5 and TRPV6 channels are expressed in blood lymphocytes are functionally active, and their activity is associated with proliferative status of blood cells.
[Show abstract][Hide abstract] ABSTRACT: Regulation of Ca(2+) entry is a key process for lymphocyte activation, cytokine synthesis and proliferation. Several members of the transient receptor potential (TRP) channel family can contribute to changes in [Ca(2+)](in); however, the properties and expression levels of these channels in human lymphocytes continue to be elusive. Here, we established and compared the expression of the most Ca(2+)-selective members of the TRPs, Ca(2+) channels transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6), in human blood lymphocytes (HBLs) and leukemia Jurkat T cells. We found that TRPV6 and TRPV5 mRNAs are expressed in both Jurkat cells and quiescent HBLs; however, the levels of mRNAs were significantly higher in malignant cells than in quiescent lymphocytes. Western blot analysis showed TRPV5/V6 proteins in Jurkat T cells and TRPV5 protein in quiescent HBLs. However, the expression of TRPV6 protein was switched off in quiescent HBLs and turned on after mitogen stimulation of the cells with phytohemagglutinin. Inwardly directed monovalent currents that displayed characteristics of TRPV5/V6 currents were recorded in both Jurkat cells and normal HBLs. In outside-out patch-clamp studies, currents were reduced by ruthenium red, a nonspecific inhibitor of TRPV5/V6 channels. In addition, ruthenium red downregulated cell-cycle progression in both activated HBLs and Jurkat cells. Thus, we identified TRPV5 and TRPV6 calcium channels, which can be considered new candidates for Ca(2+) entry into human lymphocytes. The correlation between expression of TRPV6 channels and the proliferative status of lymphocytes suggests that TRPV6 may be involved in the physiological and/or pathological proliferation of lymphocytes.
Journal of Membrane Biology 11/2012; · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Family focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis and hyalinosis of particular loops of glomeruli and is one of the causes of the nephrotic syndrome. Certain mutations in the structure of TRPC6 channels are the genetic impetus for FSGS development resulting in podocytes functional abnormalities and various nephropathies. We have recently demonstrated that non-steroid antiinflammatory drugs (NSAID) ibuprofen and diclofenac decrease the activity of endogenous TRPC-like calcium channels in the podocytes of the freshly isolated rat glomeruli. It has also been shown that TRPC6 channels are expressed in the podocytes. In the current study we have functionally reconstituted TRPC6 channels in mammalian cells to investigate the effects of diclofenac on the activity of wild type TRPC6 channel and TRPC6P112Q channel containing a mutation in the N-terminus that was described in FSGS patients. Intracellular calcium level measurements in transfected cells revealed a more intensive carbachol-induced increase of calcium concentration in HEK-293 cells expressing TRPC6P112Q versus the cells expressing wild-type TRPC6. We also performed patch-clamp experiments to study TRPC6 channels reconstituted in Chinese hamster ovary (CHO) cell line and found that application of diclofenac (500 μM) acutely reduced single channel activity. Preincubation with diclofenac (100 μM) also decreased the whole-cell current in CHO cells overexpressing TRPC6P112Q. Therefore, our previously published data on the effects of NSAID on TRPC-like channels in the isolated rat glomeruli, along with this current investigation on the cultured overexpressed mammalian cells, allows hypothesizing that TRPC6 channels may be a target for NSAID that can be important in the treatment of FSGS.
Biochemistry (Moscow) Supplement Series A Membrane and Cell Biology 07/2012; 6(3).
[Show abstract][Hide abstract] ABSTRACT: Cholesterol is a critical regulator of lipid bilayer dynamics and plasma membrane organization in eukaryotes. A variety of ion channels have been shown to be modulated by cellular cholesterol and partition into cholesterol-enriched membrane rafts. However, very little is known about functional role of membrane cholesterol in regulation of mechanically gated channels that are ubiquitously present in living cells. In our previous study, the effect of methyl-beta-cyclodextrin (MbCD), cholesterol-sequestering agent, on Ca(2+)-permeable stretch-activated cation channels (SACs) has been described. Here, cell-attached patch-clamp method was employed to search for the mechanisms of cholesterol-dependent regulation of SACs and to clarify functional contribution of lipid bilayer and submembranous cytoskeleton to channel gating. Cholesterol-depleting treatment with MbCD significantly decreased open probability of SACs whereas alpha-cyclodextrin had no effect. F-actin disassembly fully restored high level of SAC activity in cholesterol-depleted cells. Particularly, treatment with cytochalasin D or latrunculin B abrogated inhibitory effect of MbCD on stretch-activated currents. Single channel analysis and fluorescent imaging methods indicate that inhibition of SACs after cholesterol depletion is mediated via actin remodeling initiated by disruption of lipid rafts. Our data reveal a novel mechanism of channel regulation by membrane cholesterol and lipid rafts.
Biochemical and Biophysical Research Communications 08/2011; 412(1):80-5. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epithelial Na(+) channel (ENaC) activity is regulated, in part, by the cortical cytoskeleton. Here we demonstrate that cortactin is highly expressed in the kidney cortex and polarized epithelial cells, and is localized to the cortical collecting duct. Coexpression of cortactin with ENaC decreases ENaC activity, as measured in patch-clamp experiments. Biotinylation experiments and single-channel analysis reveal that cortactin decreases ENaC activity via affecting channel open probability (P(o)). Knockdown of cortactin in mpkCCD(c14) principal cells results in an increase in ENaC activity and sodium reabsorption. Coimmunoprecipitation analysis shows direct interactions between cortactin and all three ENaC subunits in cultured and native cells. To address the question of what mechanism underlies the action of cortactin on ENaC activity, we assayed the effects of various mutants of cortactin. The data show that only a cortactin mutant unable to bind Arp2/3 complex does not influence ENaC activity. Furthermore, inhibitor of the Arp2/3 complex CK-0944666 precludes the effect of cortactin. Depolymerization of the actin microfilaments and inhibition of the Arp2/3 complex does not result in the loss of association between ENaC and cortactin. Thus, these results indicate that cortactin is functionally important for ENaC activity and that Arp2/3 complex is involved in this mechanism.
The FASEB Journal 05/2011; 25(8):2688-99. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Epithelial Na(+) Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity.
First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 microg/ml; 1-2 h) or colchicine (500 microM; 1-3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD(c14) cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1-2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity.
We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.
PLoS ONE 01/2010; 5(1):e8827. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.
The FASEB Journal 06/2009; 23(9):3179-92. · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In blood cells, changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) are associated with multiple cellular events, including activation of cellular kinases and phosphatases, degranulation, regulation of cytoskeleton binding proteins, transcriptional control, and modulation of surface receptors. Although there is no doubt as to the significance of Ca(2+) signaling in blood cells, there is sparse knowledge about the molecular identities of the plasmalemmal Ca(2+) permeable channels that control Ca(2+) fluxes across the plasma membrane and mediate changes in [Ca(2+)](i) in blood cells. Using RNA expression analysis, we have shown that human leukemia K562 cells endogenously coexpress transient receptor potential vanilloid channels type 5 (TRPV5) and type 6 (TRPV6) mRNAs. Moreover, we demonstrated that TRPV5 and TRPV6 channel proteins are present in both the total lysates and the crude membrane preparations from leukemia cells. Immunoprecipitation revealed that a physical interaction between TRPV5 and TRPV6 may take place. Single-channel patch-clamp experiments demonstrated the presence of inwardly rectifying monovalent currents that displayed kinetic characteristics of unitary TRPV5 and/or TRPV6 currents and were blocked by extracellular Ca(2+) and ruthenium red. Taken together, our data strongly indicate that human myeloid leukemia cells coexpress functional TRPV5 and TRPV6 calcium channels that may interact with each other and contribute into intracellular Ca(2+) signaling.
[Show abstract][Hide abstract] ABSTRACT: The most valuable property of stem cells (SCs) is their potential to differentiate into many or all cell types of the body. So far, monitoring SC differentiation has only been possible after cells were fixed or destroyed during sample preparation. It is, however, important to develop nondestructive methods of monitoring SCs. Scanning ion conductance microscopy (SICM) is a unique imaging technique that uses similar principles to the atomic force microscope, but with a pipette for the probe. This allows scanning of the surface of living cells noninvasively and enables measurement of cellular activities under more physiological conditions than is possible with other high-resolution microscopy techniques. We report here the novel use of the SICM for studying SCs to assess and monitor the status of SCs and various cell types differentiated from SCs.
Tissue Engineering Part C Methods 01/2009; 14(4):311-8. · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transmembrane channels in blood cells are regulated by receptors, intracellular calcium stores and by different messengers involved in cell signaling. Although the calcium currents in blood cells are well studied, the molecular identity of ionic channels in these cells have not yet been determined. In recent years, a considerable progress was made in molecular and functional characterization of TRP (transient receptor potential) channels. The TRP members display a specific expression pattern, different selectivity, gating, and mode of activating and apparently provide the integration of the different signal pathways in the cells. Although the mRNAs for several types of the TRP family proteins have been found in cells of hematopoietic lineage, functional properties and the role of these plasmalemmal channels in Ca 2+ signaling in blood cells remains uncertain. Our data present the first identification of the transient receptor potential of vaniloid type 5 (TRPV5) and 6 (TRPV6) channels in K562 cells. Using RT-PCR analysis, we have shown that human leukemia K562 cells endogenously coexpress the TRPV5 and TRPV6 mRNAs. Moreover, using antiTRPV5 and anti-TRPV6 antibodies we demonstrated that TRPV5 and TRPV6 channel proteins are present both in total lysates and in crude membrane preparations from leukemia cells. Recently in plasma membrane of K562 cells we discovered the endogenous 30-pS cation channels in divalent ion-free solution (DVF). The inward rectification, voltage-dependent gating, and sensitivity to intracellular divalent ions displayed the similarity of these channels with calciumselective channels. It is known that from 29 members of the TRPs superfamily, all of them cation channels, only TRPV5 and TRPV6 exhibit properties of highly selec
Biochemistry (Moscow) Supplement Series A Membrane and Cell Biology 01/2009; 3(3):333-333.
[Show abstract][Hide abstract] ABSTRACT: This study focuses on the functional role of cellular cholesterol in the regulation of mechanosensitive cation channels activated by stretch in human leukaemia K562 cells. The patch-clamp method was employed to examine the effect of methyl-beta-cyclodextrin (MbetaCD), a synthetic cholesterol-sequestering agent, on stretch-activated single currents. We found that cholesterol-depleting treatment with MbetaCD resulted in a suppression of the activity of mechanosensitive channels without a change in the unitary conductance. The probability that the channel was open significantly decreased after treatment with MbetaCD. Fluorescent microscopy revealed F-actin reorganization, possibly involving actin assembly, after incubation of the cells with MbetaCD. We suggest that suppression of mechanosensitive channel activation in cholesterol-depleted leukaemia cells is due to F-actin rearrangement, presumably induced by lipid raft destruction. Our observations are consistent with the notion that stretch-activated cation channels in eukaryotic cells are regulated by the membrane-cytoskeleton complex rather than by tension developed purely in the lipid bilayer.
Cell Biology International 05/2007; 31(4):374-81. · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Compelling evidence shows that intracellular free magnesium [Mg(2+)](i) may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg(2+) influx in mammalian cells are poorly understood. Here, we show that mechanosensitive (MS) cationic channels activated by stretch are permeable for Mg(2+) ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl(2) when Mg(2+) was the only available carrier of inward currents. At 2 mM Mg(2+), inward mechanogated currents representing Mg(2+) influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg(2+) levels, only slight increase of single-channel currents and conductance occurred, implying that Mg(2+) permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg(2+), mean conductance values for inward currents carried by Mg(2+) were rather similar, being equal to 6.8 +/- 0.5 and 6.4 +/- 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg(2+) permeation through membrane channels activated by stretch. Our single-current measurements document Mg(2+) influx through MS channels in the plasma membrane of leukaemia cells.
Cell Research 09/2006; 16(8):723-30. · 11.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.
Journal of Cellular Physiology 01/2006; 205(3):372-8. · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly-disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca2+-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.
Biochimica et Biophysica Acta 06/2005; 1669(1):53-60. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of G proteins in regulation of non-voltage-gated Na+ channels in human myeloid leukemia K562 cells was studied by inside-out patch-clamp method. Na+ channels were activated by non-hydrolyzable analog of guanosine triphosphate (GTP), GTPgammaS, known to activate both heterotrimeric and small G proteins. Channel activity was not affected by aluminum fluoride that indiscriminately activates heterotrimeric G proteins. The effect of GTPgammaS was prevented by phalloidin and by G-actin, both interfering with actin disassembly, which indicates that GTPgammaS-induced channel activation was likely due to microfilament disruption. GTPgammaS-activated channels were inactivated by polymerizing actin. These data show, for the first time, that small G proteins can regulate Na+ channels, and an intracellular mechanism mediating their effect involves actin cytoskeleton rearrangements.