Vladislav I. Chubinskiy-Nadezhdin

Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli

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.

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.

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.

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.

Extracellular protease trypsin activates amiloride-insensitive sodium channels in human leukemia cells: SUDARIKOVA et al.

Sodium influx is tightly regulated in the cells of blood origin. Amiloride‐insensitive sodium channels were identified as one of the main sodium‐transporting pathways in leukemia cells. To date, all known regulatory pathways of these channels are coupled with intracellular actin cytoskeleton dynamics. Here, to search for physiological mechanisms controlling epithelial Na+ channel (ENaC)‐like channels, we utilized leukemia K562 cells as a unique model to examine single channel behavior in a whole‐cell patch‐clamp experiments. We have shown for the first time that extracellular serine protease trypsin directly activates sodium channels in plasma membrane of K562 cells. The whole‐cell single current recordings clearly demonstrate no inhibition of trypsin‐activated channels by amiloride or benzamil. Involvement of proteolytic cleavage in channel opening was confirmed in experiments with soybean trypsin inhibitor. More importantly, stabilization of F‐actin with intracellular phalloidin did not prevent trypsin‐induced channel activation indicating no implication of cytoskeleton rearrangements in stimulatory effect of extracellular protease. Our data reveals a novel mechanism modulating amiloride‐insensitive ENaC‐like channel activity and integral sodium permeability in leukemia cells.

Coupled Activation of Mechanosensitive and Calcium-Dependent Potassium Channels in 3T3 and 3T3-SV40 Cells

Using the patch–clamp method, mechanosensitive regulation of ion channels was studied in cultivated 3T3 and 3T3-SV40 fibroblasts. The activity of mechanosensitive cation channels with a conductivity 25 pS in response to plasma-membrane stretching was observed in both cell lines. Despite obvious differences in the actin network in normal and transformed cells, the threshold values of the stimulus required for the channel activation were close and were approximately 55 mm Hg. The frequency of channels was significantly higher in transformed 3T3-SV40 fibroblasts than in their untransformed 3T3 analogs. Coupled activation of mechanosensitive calcium-permeable channels and potassium calcium-controlled channels was found in both cell lines. The analysis of flows through single channels allows to detect functional interaction of different channels: stretch-induced local calcium entry activates potassium channels that do not have their own mechanosensitivity. The results of a comparative study show that there is a fundamental similarity between the ion mechanisms of cellular mechanotransduction in normal and transformed fibroblasts. The quantitative differences, first of all, concern the level of functional activity of mechanosensitive channels that provide the development of the local calcium signal in the near-membrane cell region.

Microfluidic chips for the study of cell migration under the effect of chemicals

Numerical simulation of the formation of a chemoattractant gradient in reaction chambers of a chip having different geometries enabled the determination of a structure suitable for the study of cell migration, in accordance with which hybrid polymer–glass microfluidic devices were manufactured. Verification of the procedures of alignment of cells in the reaction chamber of the chip by centrifugal force and subsequent culturing of the cells showed that microfluidic chips can be used to study cell migration under the effect of the chemoattractant gradient in vitro.

Role of submembranous actin cytoskeleton in regulation of non-voltage-gated sodium channels.

126 The relationship between the plasma membrane and actin cytoskeleton can ensure the integration of different signaling pathways from the cell surface to the cytoplasmic structures. It is known that the effect of a number of hormones and growth factors (includd ing the epidermal growth factor, EGF) involves rearr rangements of microfilaments [1]. It is assumed that the development of an early cell response to a stimulus strongly depends on the dynamics of the submembraa nous actin. The reorganization of the actin network affects the mechanical parameters of cells and can modulate the function of many membrane proteins, including ion channels. In solving the problems of cell signaling, an imporr tant place belongs to the physiological activation of nonnvoltageegated cation channels, which are typical for a wide range of electrically nonexcitable cells. Nonnvoltageegated sodium channels [2–6], probably belonging to the family DEG/ENaC [7, 8], were idenn tified in cells of different origin and specialization. It was previously shown [5, 9, 10] that the activity of sodium channels depends on the state of the actin cytoskeleton in human leukemia K562 cells. The purr pose of this work was to determine the functional charr acteristics and analyze the regulatory mechanisms of nonnvoltageegated sodium channels in cells with diff ferent degree of microfilament development. This is the first study to demonstrate the activation of chann nels resulting from FFactin disassembly in BALB/3T3 embryonic fibroblasts, kidney HEK293 cells, and lymphoma U937. The assembly of filaments on the membrane leads to the suppression of their activity. The results indicate an important role of the dynamics of submembranous actin in the regulation of nonnvoltt ageegated sodium channels. The patchhclamp technique was used to record ionic currents through single channels in native cell membrane fragments (celllattached variant) and in isolated membrane fragments (insideeout variant). Currents were measured using an operational amplii fier (HEKA EPC8) and analyzed as described previi ously [10, 11]. The channel activity level was estimated as the probability of the open state Р о = I/Ni, where N is the number of channels in the patch, I is the averr age current for a given time interval, and i is the averr age current through the open channel. The main external solution in the recording pipette and experii mental chamber contained 145 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES/TrisOH. Celllattached registration was performed in a …