Vedernikova Ea

Ca-dependent regulation of Na+-selective channels via actin cytoskeleton modification in leukemia cells

With the use of the patch‐clamp technique, physiological mechanisms of Na+ channel regulation involving submembranous actin rearrangements were examined in human myeloid leukemia K562 cells. We found that the actin‐severing protein gelsolin applied to cytoplasmic surface of membrane fragments at a high level of [Ca2+]i (1 μM) increased drastically the activity of Na‐selective channels of 12 pS unitary conductance. In the experiments on intact cells, the elevation of [Ca2+]i using the ionophore 4Br‐A23187 also resulted in Na+ channel activation. Addition of actin to the cytoplasmic surface of membrane patches reduced this activity to background level, likely due to actin polymerization. Our data imply that Ca‐dependent modulations of the actin cytoskeleton may represent one of the general mechanisms of channel regulation and cell signalling.

EXOGENOUS HEAT SHOCK PROTEIN HSP70 ACTIVATES POTASSIUM CHANNELS IN U937 CELLS

With the use of patch clamp technique, the effect of exogenous heat shock protein hsp70 on ion channel properties in the plasma membrane of human promonocyte U937 cells has been examined. Cell-attached experiments showed that the addition of 30-100 micrograms/ml hsp70 to the pipette solution resulted in an activation of outward currents through potassium-selective channels of 9 pS unitary conductance. The activity of K(+)-selective channels did not depend on membrane voltage and could be controlled by the intracellular free calcium concentration as revealed in inside-out recordings. K+ channels with similar conductance and kinetic behaviour were found in normal cell-attached patches very rarely. Outside-out experiments showed that the addition of hsp70 to the external solution induced a channel-like stepwise increase of inward current which may provide cation entry from the extracellular medium. The interaction of extracellular hsp70 with the membrane surface of the native cell and of the excised fragment was found to be different. The results suggest that hsp70-induced activation of Ca-dependent K channels in monocyte-macrophage cells may be due to a local increase of free Ca2+ concentration just near the inner membrane side.

Sodium Channel Activity in Leukemia Cells Is Directly Controlled by Actin Polymerization

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 μm[Ca2+] i . The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mmMgCl2 to induce actin polymerization. In the absence of MgCl2, addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.

Mechanosensitive cation channels in human leukaemia cells: calcium permeation and blocking effect

Cell‐attached and inside‐out patch‐clamp methods were employed to identify and characterize mechanosensitive (MS) ionic channels in the plasma membrane of human myeloid leukaemia K562 cells. A reversible activation of gadolinium‐blockable mechanogated currents in response to negative pressure application was found in 58 % of stable patches (n= 317). I‐V relationships measured with a sodium‐containing pipette solution showed slight inward rectification. Data analysis revealed the presence of two different populations of channels that were distinguishable by their conductance properties (17.2 ± 0.3 pS and 24.5 ± 0.5 pS), but were indistinguishable with regard to their selective and pharmacological properties. Ion‐substitution experiments indicated that MS channels in leukaemia cells were permeable to cations but not to anions and do not discriminate between Na+ and K+. The channels were fully impermeable to large organic cations such as Tris+ and N‐methyl‐d‐glucamine ions (NMDG+). Ca2+ permeation and blockade of MS channels were examined using pipettes containing different concentrations of Ca2+. In the presence of 2 mm CaCl2, when other cations were impermeant, both outward and inward single‐channel currents were observed; the I‐V relationship showed a unitary conductance of 7.7 ± 1.0 pS. The relative permeability value, PCa/PK, was equal to 0.75, as estimated at physiological Ca2+ concentrations. Partial or full inhibition of inward Ca2+ currents through MS channels was observed at higher concentrations of external Ca2+ (10 or 20 mm). No MS channels were activated when using a pipette containing 90 mm CaCl2. Monovalent mechanogated currents were not significantly affected by extracellular Ca2+ at concentrations within the physiological range (0‐2 mm), and at some higher Ca2+ concentrations.

Several types of sodium-conducting channel in human carcinoma A-431 cells

Patch clamp method in outside-out configuration was used to search for cation channels which possibly mediate sodium influx through plasma membrane in A-431 carcinoma cells. We found four types of nonvoltage-gated Na-conducting channel. The first of 9-10 pS conductance (145 mM Na+, 30 degrees C) seems to be Na-selective; three others were characterized with conductance values of 24, 35 and 65 pS and lower selectivity among cations. Na-selective channels (9-10 pS) were not blocked by tetrodotoxin (1 microM). External application of amiloride (0.1-2 mM) resulted in a reversible inhibition of single currents through Na-selective channels.

Stretch-activated ion channels in human leukemia cells

Primary signals to many cell types are mechanical. The transducers for mechanical stimuli still have not been adequately characterized, but they may include the cytoskeleton and its mechanochemical components, such as the actinand tubulin-based transporters, cell surface proteins (e.g., integrins), and ion channels [1]. Recently, there has been a resurgence of interest in the process of mechanotransduction because of a number of technical and conceptual advances. In particular, patch-clamp recording, with its inherent capability to mechanically stimulate a membrane patch while simultaneously measuring the current response [2], demonstrated the existence of a class of mechanogated membrane ion channels. Mechanogated (or mechanosensitive, MS) channels are defined by the property that their gating is determined by a membrane deformation [3]. This class appears distinct f rom voltageand l igand-gated channels [4], although recent studies indicate that mechanical stimulation can also modulate the properties of the latter channel classes [5]. For practical reasons related to the inaccessibility of specialized mechanoreceptor membranes, MS channels have been studied mostly in non-sensory cells. It was shown that MS channels are expressed in a wide range of cell types and are implicated in diverse functions, including such basic physiological processes as cell volume regulation and development. The published data have shown that MS channels form a complex class displaying different sensitivities to stimulation, gating dynamics, and open channel properties [3, 5]. However, their exact rote in many non-sensory cells remains unknown. Different properties are assumed to reflect different roles performed by the MS channels in various tissues.

Hydrogen ion block of single sodium channels in neuroblastoma cells

The effects of pH of the external medium on amplitude of currents through single sodium channels at the membrane of cultured neuroblastoma cells were investigated in mice belonging to strain C 1300, clone N18A-1. Currents through single sodium channels in isolated membrane segments (outside-out configuration) were registered with normal (7.2) and reduced (5.4) pH levels in the external medium. Reducing the pH to 5.4 was found to decrease current amplitude reversibly by about twofold (−10 to −30 mV for test potentials). Findings would confirm that the depression of macroscopic sodium currents produced by reducing the pH of the extracellular solution is due to a decline in ionic flow through single open sodium channels.