Elena A. Morachevskaya

Mechanosensitive channel activity and F-actin organization in cholesterol-depleted human leukaemia cells

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‐β‐cyclodextrin (MβCD), a synthetic cholesterol‐sequestering agent, on stretch‐activated single currents. We found that cholesterol‐depleting treatment with MβCD 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 MβCD. Fluorescent microscopy revealed F‐actin reorganization, possibly involving actin assembly, after incubation of the cells with MβCD. 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.

Cholesterol depletion-induced inhibition of stretch-activated channels is mediated via actin rearrangement

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.

Functional impact of cholesterol sequestration on actin cytoskeleton in normal and transformed fibroblasts.

Membrane cholesterol and lipid rafts are implicated in various signalling processes involving actin rearrangement in living cells. However, functional link between raft integrity and organisation of cytoskeleton remains unclear. We have compared the effect of cholesterol sequestration on F‐actin structures in normal and transformed fibroblasts in which microfilament system is developed to a different extent. The depletion of membrane cholesterol by methyl‐beta‐cyclodextrin (MbCD) resulted in a disruption of lipid rafts in plasma membrane as it was revealed by fluorescent labelling of GM1 ganglioside. In normal fibroblasts with highly developed microfilament system, the cholesterol depletion resulted in actin disassembly and reduction of stress fibres. However, in transformed cells containing low amount of fibrillar actin, MbCD treatment induced intensive formation of stress fibres and increased cell spreading. The results show that the effect of cholesterol depletion and lipid raft disruption on microfilament system is critically determined by the initial state of cytoskeleton, specifically, by the balance of polymerised and monomeric actin in the cell. We assume that uncapping of the microfilaments is the key step of cholesterol‐regulated actin remodelling.

Magnesium permeation through mechanosensitive channels: single-current measurements.

Compelling evidence shows that intracellular free magnesium [Mg2+]i may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg2+ influx in mammalian cells are poorly understood. Here, we show that mechanosensitive (MS) cationic channels activated by stretch are permeable for Mg2+ 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 MgCl2 when Mg2+ was the only available carrier of inward currents. At 2 mM Mg2+, inward mechanogated currents representing Mg2+ influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg2+ levels, only slight increase of single-channel currents and conductance occurred, implying that Mg2+ permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg2+, mean conductance values for inward currents carried by Mg2+ 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 Mg2+ permeation through membrane channels activated by stretch. Our single-current measurements document Mg2+ influx through MS channels in the plasma membrane of leukaemia cells.

Assembly of actin filaments induced by sequestration of membrane cholesterol in transformed cells

Cholesterol is a major lipid component of the plasma membrane that plays an important role in various signaling processes in mammalian cells. Our study is focused on the role of membrane cholesterol in the organization and dynamics of actin cytoskeleton. Experiments were performed on cultured transformed cells characterized by a poorly developed actin network and less prominent stress fibers: human embryonic kidney HEK293, human epidermoid larynx carcinoma HEp-2, and mouse fibroblasts 3T3-SV40. Using Factin labeling with rhodamine phalloidin, actin cytoskeleton rearrangements were analyzed after sequestration of membrane cholesterol by cyclic oligosaccharide methyl-beta-cyclodextrin and polyene macrolide antibiotic filipin. The cells treated with these agents displayed similar reorganization of actin cytoskeleton involving filament assembly. In HEp-2 carcinoma cells and 3T3-SV40 fibroblasts, cholesterol-sequestering reagents induced intense stress fiber formation and enhanced cell spreading; i.e., features of transformed phenotype reversion were observed. The cytoskeleton rearrangements are probably initiated by disruption of lipid raft integrity that is critically dependent on the level of the membrane cholesterol.

Non-hydrolyzable analog of GTP induces activity of Na+ channels via disassembly of cortical actin cytoskeleton

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), GTPγS, 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 GTPγS was prevented by phalloidin and by G‐actin, both interfering with actin disassembly, which indicates that GTPγS‐induced channel activation was likely due to microfilament disruption. GTPγS‐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.

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca(2+)-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca(2+) entry mediated by SACs presumably has a significant impact on various Ca(2+)-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 Ca(2+) influx via SACs triggered the activity of Ca(2+)-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 Ca(2+) concentration due to SAC activity may provide a functional link between various Ca(2+)-dependent molecules in the processes of cellular mechanotransduction.

Local calcium signalling is mediated by mechanosensitive ion channels in mesenchymal stem cells

Mechanical forces are implicated in key physiological processes in stem cells, including proliferation, differentiation and lineage switching. To date, there is an evident lack of understanding of how external mechanical cues are coupled with calcium signalling in stem cells. Mechanical reactions are of particular interest in adult mesenchymal stem cells because of their promising potential for use in tissue remodelling and clinical therapy. Here, single channel patch-clamp technique was employed to search for cation channels involved in mechanosensitivity in mesenchymal endometrial-derived stem cells (hMESCs). Functional expression of native mechanosensitive stretch-activated channels (SACs) and calcium-sensitive potassium channels of different conductances in hMESCs was shown. Single current analysis of stretch-induced channel activity revealed functional coupling of SACs and BK channels in plasma membrane. The combination of cell-attached and inside-out experiments have indicated that highly localized Ca2+ entry via SACs triggers BK channel activity. At the same time, SK channels are not coupled with SACs despite of high calcium sensitivity as compared to BK. Our data demonstrate novel mechanism controlling BK channel activity in native cells. We conclude that SACs and BK channels are clusterized in functional mechanosensitive domains in the plasma membrane of hMESCs. Co-clustering of ion channels may significantly contribute to mechano-dependent calcium signalling in stem cells.

Molecular and functional identification of sodium channels in K562 cells

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various nonexcitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na+ channels (DEG/ENaC) can be considered as possible molecular correlates. By using RT-PCR, expression of α-, β-, and γ-hENaC subunits in the K562 cells was detected. Various modes of the patch-clamp method were used to examine functional properties of sodium channels—specifically, to test the effect of amiloride on single channel and integral currents. The biophysical characteristics of the NVSG channels were close to those of ENaC; the channels have unitary conductance of 12 pS (145 mM Na+) and were impermeable to divalent cations (Ca2+ and Mg2+). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in the K562 cells belong to the ENaC family.

Simvastatin induced actin cytoskeleton disassembly in normal and transformed fibroblasts without affecting lipid raft integrity

Statins are the most commonly prescribed agents used to modulate cholesterol levels in course of hypercholesterolemia treatment because of their relative tolerability and LDL‐C lowering effect. Recently, there are emerging interests in the perspectives of statin drugs as anticancer agents based on preclinical evidence of their antiproliferative, proapoptotic, and anti‐invasive properties. Functional impact of statin application on transformed cells still remains obscure that requires systematic study on adequate cellular models to provide correct comparison with their non‐transformed counterparts. Cholesterol is the major lipid component of mammalian cells and it plays a crucial role in organization, lateral heterogeneity, and dynamics of plasma membrane as well as in membrane‐cytoskeleton interrelations. To date, it is uncertain whether cellular effects of statins involve lipid‐dependent alteration of plasma membrane. Here, the effects of simvastatin on lipid rafts, F‐actin network and cellular viability were determined in comparative experiments on transformed fibroblasts and their non‐transformed counterpart. GM1 lipid raft marker staining indicated no change of lipid raft integrity after short‐ or long‐term simvastatin treatments. In the same time, simvastatin induced cytoskeleton rearrangement including partial F‐actin disruption in cholesterol‐ and lipid raft‐independent manner. Simvastatin dose‐dependently affected viability of BALB/3T3 and 3T3B‐SV40 cell lines: transformed fibroblasts were noticeably more sensitive to simvastatin comparing to non‐transformed cells.