Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling.

Video microscopy and whole-cell patch-clamp recording were used to monitor changes in relative cell volume (V/Vo), chloride conductance (gCl), and membrane capacitance (Cm) during osmotically induced swelling in Jurkat T lymphocytes. Cellular swelling was initiated with hyperosmotic pipette solutions. Simultaneous evaluation of V/Vo and gCl revealed a 59-s delay between the inception of swelling and the activation of outwardly rectifying, ATP-dependent Cl- channels. Following the delay, increases in V/Vo and gCl progressed in parallel. In contrast, Cm, a measure of cell surface area, fell gradually at a rate of approximately 150 fF/min after whole-cell access was achieved. The decline in Cm lasted 200 s and was followed by a rapid rise (approximately 750 fF/min). The rise in Cm coincided with a variable increase in "leak" current, gCl increased at a slower rate and reached lower peak values in experiments performed without ATP; ATP had no effect on the biphasic Cm time course. The temporal separation of conductance and capacitance during swelling suggests that gCl and Cm vary independently, supporting the hypothesis that a large portion, if not all, of the whole-cell Cl- conductance activated during swelling is provided by volume-sensitive Cl- channels preexisting in the plasma membrane.     

Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling-induced activation of hIK channels was strongly inhibited by cytochalasin D (CD), in concentrations that caused depolymerization of F-actin filaments, indicating a role for the F-actin cytoskeleton in modulation of hIK by changes in cell volume. In conclusion, hIK and rSK3 channels are activated by cell swelling and inhibited by shrinkage. A role for the F-actin cytoskeleton in the swelling-induced activation of hIK channels is suggested.     

Enhancement of conductive anion permeability in cultured cells by cetiedil

Cetiedil, a drug that is reported to block K+-channels, substantially increases the conductive C1- permeability of Chinese hamster ovary (CHO) cells. The permeability was monitored by volume changes in cells treated with gramicidin to increase the cation permeability. Under this circumstance, increases in Cl- conductances result in volume changes detectable by electronic sizing, with the direction determined by the gradients of the permeating ions. In NaCl or KCl media, swelling occurs, but in N-methylglucamine chloride, shrinking. The increases in Cl- conductance could also be measured as an increased 36Cl- flux or by changes in membrane potential (measured by fluorescence of a potential-sensitive dye) toward the Cl- equilibrium potential. The effect of cetiedil was concentration dependent, with maximal effect at 50 microM. The anion specificity for the conductance was NO3- greater than Cl- = Br- much greater than SO4-2 or isethionate. A number of other drugs that influence transport activities had no effect on Cl- conductance. The cetiedil effect on Cl- conductance was observed in one other cell line, but was absent in several other cell types. The cetiedil-induced Cl- conductance in CHO cells appears to involve a different pathway than that induced by exposure to hypotonic medium.     

Role of water channels in the regulation of the volume of principal cells of rat kidney collecting ducts in hypoosmotic medium

The effect of plasma membrane water permeability on the rate of changes in the volume of principal cells of collecting ducts of the outer substantia medullaris under conditions of hypoosmotic shock has been studied. Changes in cell volume were studied by the fluorescent method. It was shown that the hypotonic shock induced a rapid increase in the cell volume with the characteristic time that depended on plasma membrane water permeability. The decrease in volume occurred much more slowly, and the rate of volume decrease directly correlated with the rate of swelling. The inhibition of potassium transport by barium chloride decreased the rate of volume restoration, without affecting substantially the duration of the swelling phase. The inhibition of mercury-sensitive water channels by mercury caused a significant increase in the time of both cell swelling and volume restoration. It was concluded that the state of water channels largely determines the rate of the regulatory response of epithelial cells of collecting ducts to hypoosmotic shock and affects the exchange of cell osmolites.     

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Receptor-dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface-bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl- gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside-out and cell-attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor-independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.     

Swelling activates chloride current and increases internal calcium in nonpigmented epithelial cells from the rabbit ciliary body

Membrane current and [Ca]_i in rabbit nonpigmented ciliary body epithelial cells (NPE cells) were monitored with combined patch-clamp and fura-2 measurements during cell swelling induced by anisosmotic conditions. In the presence of K-channel blockers, cell swelling produced an increase in membrane current, accompanied by an increase in [Ca]_i. Structural changes in the cell, associated with membrane deformation, may be the cause of the increase in [Ca]_i during swelling. The conductance activated by swelling was permeable to CI: it was dependent on the CI concentration gradient across the cell membrane, and it was blocked by the CI-channel blockers DIDS, SITS, NPPB, and DIOA. Although swelling increased both CI current and [Ca]_i, there was no evidence that Ca was involved in the regulation of the CI conductance. Cell swelling activated the current even when [Ca]_i was strongly buffered at an elevated level (500 nM) or at a low level (～0) with internal Ca-BAPTA/Cs-BAPTA mixtures. In addition, CI conductance was unaffected when [Ca]_i was increased with a Ca ionophore. There was also no evidence that cAMP participates in the regulation of the CI conductance: swelling activation of the current occurred in the presence of cAMP inhibitor (R_p-cAMP-S) and cAMP mimic (S_p-cAMP-S). The data suggest independent involvement of CI conductance and internal Ca in the regulation of cell volume in NPE cells. © 1995 Wiley-Liss, Inc.     

Ionic mechanisms of cardiac cell swelling induced by blocking Na+/K+ pump as revealed by experiments and simulation

Although the Na(+)/K(+) pump is one of the key mechanisms responsible for maintaining cell volume, we have observed experimentally that cell volume remained almost constant during 90 min exposure of guinea pig ventricular myocytes to ouabain. Simulation of this finding using a comprehensive cardiac cell model (Kyoto model incorporating Cl(-) and water fluxes) predicted roles for the plasma membrane Ca(2+)-ATPase (PMCA) and Na(+)/Ca(2+) exchanger, in addition to low membrane permeabilities for Na(+) and Cl(-), in maintaining cell volume. PMCA might help maintain the [Ca(2+)] gradient across the membrane though compromised, and thereby promote reverse Na(+)/Ca(2+) exchange stimulated by the increased [Na(+)](i) as well as the membrane depolarization. Na(+) extrusion via Na(+)/Ca(2+) exchange delayed cell swelling during Na(+)/K(+) pump block. Supporting these model predictions, we observed ventricular cell swelling after blocking Na(+)/Ca(2+) exchange with KB-R7943 or SEA0400 in the presence of ouabain. When Cl(-) conductance via the cystic fibrosis transmembrane conductance regulator (CFTR) was activated with isoproterenol during the ouabain treatment, cells showed an initial shrinkage to 94.2 +/- 0.5%, followed by a marked swelling 52.0 +/- 4.9 min after drug application. Concomitantly with the onset of swelling, a rapid jump of membrane potential was observed. These experimental observations could be reproduced well by the model simulations. Namely, the Cl(-) efflux via CFTR accompanied by a concomitant cation efflux caused the initial volume decrease. Then, the gradual membrane depolarization induced by the Na(+)/K(+) pump block activated the window current of the L-type Ca(2+) current, which increased [Ca(2+)](i). Finally, the activation of Ca(2+)-dependent cation conductance induced the jump of membrane potential, and the rapid accumulation of intracellular Na(+) accompanied by the Cl(-) influx via CFTR, resulting in the cell swelling. The pivotal role of L-type Ca(2+) channels predicted in the simulation was demonstrated in experiments, where blocking Ca(2+) channels resulted in a much delayed cell swelling.     

Differentiation of two distinct K conductances in the basolateral membrane of turtle colon

The K conductance of the basolateral membrane of turtle colon was measured in amphotericin-treated cell layers under a variety of ionic conditions. Changing the composition of the bathing solutions changed not only the magnitude but also the physical properties of the basolateral K conductance. The results are consistent with the notion that altered ionic environments can lead to changes in the relative abundance of two different populations of K channels in the basolateral membrane, which can be differentiated on the basis of pharmacological specificity, ion selectivity, and tracer kinetics. In the following article (Germann, W. J., S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:253-274), we present evidence consistent with the hypothesis that one of these conductances was due to the same channels that give rise to the normal resting basolateral K conductance of the transporting cells, while the other was associated with experimental maneuvers that led to extreme swelling of the epithelial cells.     

Calcium-independent cell volume regulation in human lymphocytes. Inhibition by charybdotoxin

The properties of the K+ pathway underlying regulatory volume decrease (RVD) in human blood lymphocytes were investigated. Evidence is presented for the existence of three types of K+ conductance in these cells. Ionomycin, a Ca2+ ionophore, induced a K(+)-dependent hyperpolarization, indicating the presence of Ca2(+)-activated K+ channels, which were blocked by charybdotoxin (CTX). CTX also induced a depolarization of the resting membrane potential, even at subphysiological cytosolic [Ca2+]([Ca2+]i), which suggests the existence of a second CTX-sensitive, but Ca2(+)-independent conductance. A CTX-resistant K+ conductance was also detected. RVD in blood lymphocytes was partially (approximately 75%) blocked by CTX. However, volume regulation was not accompanied by detectable changes in [Ca2+]i, nor was it prevented by removal of extracellular Ca2+ and depletion or buffering of intracellular Ca2+. These observations suggest that K+ loss during RVD is mediated by Ca2(+)-independent, CTX-sensitive channels or that Ca2(+)-dependent channels can be activated by cell swelling at normal or subnormal [Ca2+]i. The former interpretation is supported by findings in rat thymic lymphocytes. These cells also displayed a CTX-sensitive Ca2(+)-dependent hyperpolarization. However, CTX did not significantly alter the resting potential, suggesting the absence of functional Ca2(+)-independent, toxin-sensitive channels. Volume regulation in thymic lymphocytes was less efficient than in human blood cells. In contrast to blood lymphocytes, RVD in thymocytes was not affected by CTX. These observations indicate that, though present in lymphocytes, Ca2(+)-activated K+ channels do not play an important role in volume regulation. Instead, RVD seems to be mediated by Ca2(+)-independent K+ channels. We propose that two types of channels, one CTX sensitive and the other CTX insensitive, mediate RVD in human blood lymphocytes, whereas only the latter type is involved in rat thymocytes.     

Ca2+-activated IK1 channels associate with lipid rafts upon cell swelling and mediate volume recovery

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca2+-dependent activation of K+ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K+-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K+ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.     

Regulation of potassium conductance in the cellular membrane at early embryogenesis.

At the early stages of development of the fresh water fish loach (Misgurnus fossilis) the resting membrane potential (Er) of cleaving cells oscillates periodically with an amplitude of 8-12 mV. Er oscillation correlates with the cell cycle and is accompanied by changes of K+ conductivity. Two types of K(+)-selective ionic channels with conductance of approximately 70 and 25 pS in symmetrical (150 mM KCl) solution were observed in the membrane of cleaving loach embryos. 'High' conductance and 'low' conductance channels were recorded in approximately 90% and 10% of patches investigated (n = 275), respectively? The activity of 'high' conductance channels was regulated by the application of pressure to the membrane, ie these channels were stretch-activated (SA). The activity of SA channels changes dramatically during the cell-cleavage cycle. At the beginning of interphase the probability of SA channels being in the open state (P0) was minimal, while at prometaphase the probability was increased 10-100-fold. Application of ATP to the cytoplasmic inside-out patches induced a reversible elevation of stretch sensitivity of the SA channels in 50% of the patches, while the non-hydrolyzable analogue of ATP was not effective. Combined application of ATP, cAMP and cAMP-dependent protein kinase (PK) induced a reversible elevation in the SA channel activity while inhibitors of PK prevented its activating effects. Phosphatase inhibitors prolonged the activating effect of PK on SA channels. We propose that oscillations of the resting potential during the cell-cleavage cycle arise due to modulation of SA channel sensitivity to stretch through cAMP-dependent phosphorylation.     

Activation of K+ and Cl− channels in MDCK cells during volume regulation in hypotonic media

Single-channel patch-clamp experiments were performed on MDCK cells in order to characterize the ionic channels participating in regulatory volume decrease (RVD). Subconfluent layers of cultured cells were exposed to a hypotonic medium (150 mOsm), and the membrane currents at the single-channel level were measured in cell-attached experiments. The results indicate that MDCK cells respond to a hypotonic swelling by activating several different ionic conductances. In particular, a potassium and a chloride channel appeared in the recordings more frequently than other channels, and this allowed a more detailed study of their properties in the inside-out configuration of the patch-clamp technique. The potassium channel had a linear I/V curve with a unitary conductance of 24 +/- 4 pS in symmetrical K+ concentrations (145 mM). It was highly selective for K+ ions vs. Na+ ions: PNa/PK less than 0.04. The time course of its open probability (P0) showed that the cells responded to the hypotonic shock with a rapid activation of this channel. This state of high activity was maintained during the first minute of hypotonicity. The chloride channel participating in RVD was an outward-rectifying channel: outward slope conductance of 63.3 +/- 4.7 pS and inward slope conductance of 26.1 +/- 4.9 pS. It was permeable to both Cl- and NO3- and its maximal activation after the hypotonic shock was reached after several seconds (between 30 and 100 sec). The activity of this anionic channel did not depend on cytoplasmic calcium concentration. Quinine acted as a rapid blocker of both channels when applied to the cytoplasmic side of the membrane. In both cases, 1 mM quinine reversibly reduced single-channel current amplitudes by 20 to 30%. These results indicate that MDCK cells responded to a hypotonic swelling by an early activation of highly selective potassium conductances and a delayed activation of anionic conductances. These data are in good agreement with the changes of membrane potential measured during RVD.     

Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane

Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of -41 mV (SD 16) and -112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.     

Resting and osmotically induced basolateral K conductances in turtle colon

Two types of K conductance can be distinguished in the basolateral membranes of polyene-treated colonic epithelial cells (see Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:237-251). The significance of these two types of K conductance was investigated by measuring the properties of the basolateral membrane under conditions that we presumed would lead to marked swelling of the epithelial cells. We compared the basolateral conductance under these conditions of osmotic stress with those observed under other conditions where changes in cell volume would be expected to be less dramatic. In the presence of a permeant salt (KCl) or nonelectrolyte (urea), amphotericin-treated colonic cell layers exhibited a quinidine-sensitive conductance. Light microscopy revealed that these conditions were also associated with pronounced swelling of the epithelial cells. Incubation of tissues in solutions containing the organic anion benzene sulfonate led to the activation of the quinidine-sensitive gK and was also associated with dramatic cell swelling. In contrast, tissues incubated with an impermeant salt (K-gluconate) or nonelectrolyte (sucrose) did not exhibit a quinidine-sensitive basolateral conductance in the presence of the polyene. Although such conditions were also associated with changes in cell volume, they did not lead to the extreme cell swelling detected under conditions that activated the quinidine-sensitive gK. The quinidine-sensitive basolateral conductance that was activated under conditions of osmotic stress was also highly selective for K over Rb, in contrast to the behavior of normal Na transport by the tissue, which was supported equally well by K or Rb and was relatively insensitive to quinidine. The results are consistent with the notion that the basolateral K conductance measured in the amphotericin-treated epithelium bathed by mucosal K-gluconate solutions or in the presence of sucrose was due to the same channels that are responsible for the basolateral K conductance under conditions of normal transport. Conditions of extreme osmotic stress, however, which led to pronounced swelling of the epithelial cells, were associated with the activation of a new conductance, which was highly selective for K over Rb and was blocked by quinidine or lidocaine.     

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Kinetics of voltage-induced conductance increases in the outer mitochondrial membrane.

The kinetics of the increase in conductance in the outer mitochondrial membrane induced by patch-clamping at various negative potentials (pipette inside negative) are reported. The changes are biphasic, a rapid increase is followed by a slowly developing larger change. The results can be predicted by a model in which an initial activation of channels is followed by their assembly into highly conducting channels. The model suggests that five to seven activated subunits form each high-conductance channel.     

Changes in membrane cholesterol of pituitary tumor (GH3) cells regulate the activity of large-conductance Ca2+-activated K+ channels

The effects of changes in membrane cholesterol on ion currents were investigated in pituitary GH3 cells. Depletion of membrane cholesterol by exposing cells to methyl-beta-cyclodextrin (MbetaCD), an oligosaccharide, resulted in an increase in the density of Ca2+-activated K+ current (IK(Ca)). However, no significant change in IK(Ca) density was demonstrated in GH3 cells treated with a mixture of MbetaCD and cholesterol. Cholesterol depletion with MbetaCD (1.5 mg/ml) slightly suppressed the density of voltage-dependent L-type Ca2+ current. In inside-out patches recorded from MbetaCD-treated cells, the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels was enhanced with no change in single-channel conductance. In MbetaCD-treated cells, voltage-sensitivity of BK(Ca) channels was increased; however, no change in Ca2+-sensitivity could be demonstrated. A negative correlation between adjacent closed and open times in BK(Ca) channels was observed in MbetaCD-treated cells. In inside-out patches from MbetaCD-treated cells, dexamethasone (30 microM) applied to the intracellular surface did not increase BK(Ca)-channel activity, although caffeic acid phenethyl ester and cilostazol still opened its probability effectively. However, no modification in the activity of ATP-sensitive K+ channels could be seen in MbetaCD-treated cells. Current-clamp recordings demonstrated that the cholesterol depletion maneuver with MbetaCD reduced the firing of action potentials. Therefore, the increase in BK(Ca)-channel activity induced by membrane depletion may influence the functional activities of neurons or neuroendocrine cells if similar results occur in vivo.     

Transduction of membrane tension by the ion channel alamethicin.

Mechanoelectrical transduction in biological cells is generally attributed to tension-sensitive ion channels, but their mechanisms and physiology remain controversial due to the elusiveness of the channel proteins and potential cytoskeletal interactions. Our discovery of membrane tension sensitivity in ion channels formed by the protein alamethicin reconstituted into pure lipid membranes has demonstrated two simple physical mechanisms of cytoskeleton-independent transduction. Single channel analysis has shown that membrane tension energizes mechanical work for changes of conductance state equal to tension times the associated increase in membrane area. Results show a approximately 40 A2 increase in pore area and transfer of an 80-A2 polypeptide into the membrane. Both mechanisms may be implicated in mechanical signal transduction by cells.