With the use of the patch clamp technique, the role of cytoskeleton in the regulation of ion channels in plasma membrane of leukemic K562 cells was examined. Single-channel measurements have indicated that disruption of actin filaments with cytochalasin D (CD) resulted in a considerable increase of the activity of non-voltage-gated sodium-permeable channels of 12 pS unitary conductance. Background activity of these channels was low; open probability (po) did not exceed 0.01-0.02. After CD, po grew at least 10-20 times. Cell-attached and whole-cell recordings showed that activation of sodium channels was elicited within 1-3 min after the addition of 10-20 micrograms/ml CD to the bath extracellular solution or in the presence of 5 micrograms/ml CD in the intracellular pipette solution. Preincubation of K562 cells with CD during 1 h also increased drastically the activity of 12 pS sodium channels. Whole-cell measurements confirmed that CD-activated channels were permeable to monovalent cations (preferentially to Na+ and Li+), but not to bivalent cations (Ca2+, Ba2+). Colchicine (1 microM), which affect microtubules, did not alter background channel activity. Our data indicate that actin filaments organization plays an important role in the regulation of sodium-permeable channels which may participate in providing passive Na+ influx in red blood cells.

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in nonexcitable 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 M [Ca 2؉ ] 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 mM MgCl 2 to induce actin polymerization. In the absence of MgCl 2 , 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.Functional coupling between channel proteins and the cortical cytoskeleton may play a key role in membrane ion transport and cellular signaling. Involvement of F-actin in ion channel functioning has been established and studied extensively in polarized epithelial cells (1-7). Specifically, several lines of evidence revealed an association between the amiloride-sensitive sodium channels and the actin-based cytoskeleton in renal epithelia. Indirect immunofluorescence and confocal microscopy demonstrated that sodium channels in the apical membrane colocalize with actin, spectrin (fodrin), and ankyrin (5, 6). Electrophysiological studies on epithelial A6 cells showed that disruption of actin microfilament networks by cytochalasin D induced sodium channel activity both in cell-attached and excised patches (1). Similar effects were observed in the presence of actin or actin-gelsolin complexes added to the cytoplasmic side of excised inside-out patches, whereas the actin-DNase I complexes did not activate sodium channels. These results were explained by a model suggesting that the channels are activated by short actin filaments produced either by severing of endogenous long filaments with cytochalasin or by assembly from monomeric actin during spontaneous or gelsolin-mediated polymerization (1). Similar observations were made on planar lipid bilayers containing cloned epithelial sodium channels (4, 7). In addition, interaction of actin with epithelial channels was reported to modulate considerably the intrinsic...

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 [Ca 2+ h (1 /xM) increased drastically the activity of Na-selective channels of 12 pS unitary conductance. In the experiments on intact cells, the elevation of [Ca 2+ ]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 Cadependent modulations of the actin cytoskeleton may represent one of the general mechanisms of channel regulation and cell signalling.

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.

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.