Extracellular calcium affects the membrane currents of cultured human keratinocytes.

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage-clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of -24 mV. Voltage-clamp experiments showed that these cells had two membrane ionic currents: a large voltage-independent leak conductance, and a smaller voltage-dependent Cl- current that activated with depolarization. Increasing the extracellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage-gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2(+)-activated Cl- current in these cells suggests a role for this conductance in the initiation of differentiation.     

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.     

Depolarization of macrophage polykaryons in the absence of external sodium induces a cyclic stimulation of a calcium-activated potassium conductance

Macrophage polykaryons associated with the foreign body granuloma display several electrophysiological properties when studied with intracellular microelectrodes. One of the most evident properties is the slow hyperpolarization (2-5 s long, 10-60 mV amplitude), due to transient openings of Ca2+-dependent K+ channels, that is similar to those observed in macrophages. How this oscillation of membrane potential is triggered is not well known and the only way to repeatedly activate it under experimental control is through the intracellular injection of Ca2+. Although this technique is important for understanding the properties of the K+ channels, no information has been obtained about the way Ca2+ levels are raised and controlled in the cytosol. Slow hyperpolarizations can also be triggered by electrical stimulation, but reproducibility is low with cells bathed in physiological solutions. We then decided to investigate the effect of depolarization on the electrophysiological properties of macrophage polykaryons exposed to bathing solutions of several ionic compositions. We show in this paper that cell membrane depolarization induced by a long current pulse can trigger several patterns of membrane potential changes and that, in the absence of extracellular Na+, repetitive oscillations of decaying amplitudes are observed in almost all the cells. They are very similar to the slow hyperpolarizations, are dependent on the presence of extracellular Ca2+, and are blocked by quinine and D-600. Whole-cell patch clamp recording under voltage clamp conditions showed an outward current that oscillates and that also exhibits decaying amplitudes. The data presented here indicate that these oscillations are a consequence of the cyclic opening of the Ca2+-activated K+ channels and support the hypothesis that favors the participation of Ca2+ channels and of the Ca2+/Na+ exchange system in their triggering. These two mechanisms are not enough to explain either why the K+ channels close or why the membrane potential returns to the original level at the end of each cycle. The possibility of using these oscillations as a model to study the slow hyperpolarization is discussed.     

Commitment to differentiation of murine erythroleukemia cells involves a modulated plasma membrane depolarization through Ca2+-activated K+ channels

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.     

Bending the Primary Cilium Opens Ca2+-sensitive Intermediate-Conductance K+ Channels in MDCK Cells

Increasing tubular fluid flow rate has previously been shown to induce K+ secretion in mammalian cortical collecting duct. The mechanism responsible was examined in the present study using MDCK cells as a model. The change in membrane potential difference (EM) of MDCK cells was measured with a fluorescent voltage-sensitive dye, DiBAC4(3), when the cell's primary cilium was continuously bent with a micropipette or by the flow of perfusate. Bending the cilium produced a hyperpolarization of the membrane that lagged behind the increase in intracellular Ca2+ concentration by an average of 36 seconds. Gd3+, an inhibitor of the flow-induced Ca2+ increase, prevented the hyperpolarization. Blocking K+ channels with Ba2+ reduced the flow-induced hyperpolarization, implying that it resulted from activation of Ca2+-sensitive K+ channels. Further studies demonstrated that the hyperpolarization was diminished by the blocker of Ca2+-activated K+ channels, charybdotoxin, whereas iberiotoxin or apamin had no effect, results consistent with the activation of intermediate-conductance Ca2+-sensitive K+ channels. RT-PCR analysis and sequencing confirmed the presence of intermediate-conductance K+ channels in MDCK cells. We conclude that the increase in intracellular Ca2+ associated with bending of the primary cilium is the cause of the hyperpolarization and increased K+ conductance in MDCK cells.     

Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca2+-activated K+ (BK(Ca)) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK(Ca) channels opener NS1619 reversed thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization; BK(Ca) channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca2+ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK(Ca) channels may play a regulatory role in it.     

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.     

hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation.

Human T lymphocytes express a Ca2+-activated K+ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca2+-binding sites; however, we mapped a Ca2+-dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (Vm) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (Vm approximately 0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological Vm values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.     

The relationship between mitogen-induced membrane potential changes and intracellular free calcium in human T-lymphocytes

We have investigated the effects of mitogenic lectins on human T-lymphocytes, isolated from peripheral blood, and cells from the T-cell clone, HPB-ALL, using the fluorescent dyes, bis-thiobarbiturate tri-methineoxonol (bisoxonol) and quin2 to sense changes in membrane potential and intracellular free [Ca2+], respectively. The resting potential of both cell types is close to the K+ equilibrium potential. Changes from the resting level occur when mitogenic concentrations of either concanavalin A or phytohaemagglutinin are added. T-lymphocytes undergo a decrease in emission, maximal at 1 to 2 min, corresponding to a small membrane hyperpolarization. This is followed by a depolarization to approximately the resting level. HPB-ALL cells, on the other hand, respond to the mitogens by a sustained increase in fluorescence, denoting a depolarization, that is maximal at 4 to 5 min and 7 to 9 min, respectively. The Ca2+-dependence of these phenomena indicates that the membrane potential response, in both cell types, is the resultant of two opposing effects: a Ca2+-sensitive ion movement tending to hyperpolarize the cells and a Ca2+-insensitive effect that generates a depolarization. Our results suggest that Ca2+-activated K+ channels are responsible for the first effect and that an inward Na+ movement accounts for the depolarization signal in T-lymphocytes. In HPB-ALL cells only part of the depolarization is Na+-dependent. Although the effects elicited by phytohaemagglutinin occur more slowly than those produced by concanavalin A, similar membrane potential and [Ca2+]i changes occur.     

Transient and sustained effects of hormones and calcium on membrane potential in a bone cell clone

Measurements were made of the electrophysiological and cAMP response to changes in extracellular [Ca2+] and to hormone application in a bone cell clone. Both transient and long-term electrophysiological responses were studied. An increase in extracellular [Ca2+] usually resulted in a transient hyperpolarization of about 60-sec duration. In addition, increases in extracellular [Ca2+] from 0.9 to 1.8 mM and from 1.8 to 3.6 mM resulted in long-term hyperpolarization and increased potential fluctuations. Increasing bathing [Ca2+] until the membrane potential reached the K+ equilibrium level resulted in a significant decrease in fluctuations. Addition to the bathing medium of quinine, a putative blocker of the Ca2+-dependent K+ channel, resulted in long-term depolarization of the mean membrane potential, and a long-term decrease in potential fluctuations. Addition of Mg2+, a mild antagonist of Ca2+ entry into the cell, produced transient depolarization and reduction of potential fluctuations. These effects suggest that the potential fluctuations reflect cytoplasmic [Ca2+] fluctuations via Ca2+-dependent K+ membrane channels. Under an extracellular [Ca2+] of 1.8 mM, the application of prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone produced no significant effect on mean membrane potential or on the sustained potential fluctuations, but PGE2 did significantly raise intracellular cAMP. Under an increased bathing [Ca2+], significant changes in mean potential and fluctuations did occur in response to PGE2, but not in response to the other hormones, while the PGE2 effect on cAMP was not greatly changed. Hyperpolarizing transients of about 30-sec duration occurred in response to all of the hormones, particularly at an extracellular [Ca2+] of 3.6 mM. Thus, there are both transient and long-term electrophysiological responses to hormone application, with only the long-term response correlated with the production of cAMP. These electrophysiological responses may represent separate transient and long-term calcium transport responses to hormone application.     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Voltage gating of Ca2(+)-activated potassium channels in human lymphocytes

The effect of membrane potential on Ca2+ activated K+ channels was studied on human peripheral lymphocytes. Membrane potential was monitored using bisoxonol and flow cytometry. 1 mM Ca2+ in the presence of 2 microM ionomycin depolarized the control cell population, while 100 microM Ca2+ caused hyperpolarization. However 1 mM Ca2+ had a hyperpolarizing effect on previously partially depolarized cells. Potassium channel blockers did not influence the depolarization, while they inhibited the hyperpolarization. Based on the experimental evidence a voltage gating of Ca2+ activated K+ channels is suggested.     

Digitonin-permeabilized colonic cell layers. Demonstration of calcium- activated basolateral K+ and Cl- conductances

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.     

Role of Ca2+-activated Cl- channels in the mechanism of apoptosis induced by cyclosporin A in a human hepatoma cell line

The mechanism of apoptosis induced by cyclosporin A (CsA) in a human hepatoma cell line was investigated. CsA induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoma cells. CsA induced Cl- efflux, which was significantly blocked by niflumic acid (NA), a specific inhibitor, and flufenamic acid (FA), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), and 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), non-specific inhibitors of Ca2+-activated Cl- channels (CaCCs), not by calyculin A, an inhibitor of K+,Cl- -cotransport. In addition, CsA did not alter intracellular K+ concentration. Moreover, CsA increased intracellular Ca2+ concentration, and treatment with BAPTA/AM, an intracellular Ca2+ chelator, significantly inhibited the CsA-induced Cl- efflux, indicating that CsA induced Cl- efflux through the activation of CaCCs. Treatment with these CaCC inhibitors (NA, FA, NPPB, and DIDS) markedly prevented the CsA-induced apoptosis. Taken together, these results suggest that CaCCs may mediate apoptosis induced by CsA in HepG2 cells. Furthermore, these results provide a new insight into the novel function of CaCCs in the regulation of cancer cell apoptosis associated with perturbation of intracellular Ca2+ signal.     

Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.     

Intracellular Mg2+ increases neuronal excitability.

Injection of Mg2+ into spinal motoneurons of cats leads to a depolarization, associated with a fall in membrane conductance, diminution in post-spike hyperpolarization, and increased excitability. This action has an apparent reversal level substantially more negative than the resting potential, and can be ascribed to a fall in K+ membrane conductance. Since these effects are opposite to those produced by intracellular Ca2+, it is suggested that Mg2+ probably competes with Ca2+ at the Ca2+-activated K+ ionophoreal free ionophores. Neuronal excitability can be regulated by the ratio of internal free Ca2+/Mg2+.     

The effects of ionophore A23187 and concanavalin A on the membrane potential of human peripheral blood lymphocytes and rat thymocytes

Effects of the Ca2+-ionophore A23187 and concanavalin A on the membrane potential of human lymphocytes and rat thymocytes have been studied using the fluorescent potential probe diS-C3-(5). At concentrations of 10(-8) to 10(-6) M A23187 changes the membrane potential, inducing both hyper- and depolarization. Depending on concentrations of A23187 and the external Ca2+, and on the type of lymphocytes, one of these effects predominates. The hyperpolarization induced by A23187 is caused by activation of Ca2+-dependent K+ channels. It is blocked by quinine and high concentrations of extracellular K+. The dependence of Ca2+-activated K+ transport on extracellular Ca2+ and its sensitivity to calmodulin antagonists is different for human lymphocytes and for thymocytes. As distinct from lymphocytes, in thymocytes calmodulin is not involved in activation of Ca2+-dependent K+ transport. The depolarization induced in lymphocytes by A23187 is caused by an increase in Na+ permeability of the lymphocyte plasma membrane: it is eliminated in a low-Na+ medium. At mitogenic concentrations concanavalin A does not change the membrane potential of the lymphocytes. The results obtained permit elucidation of the relationship between two early events in lymphocyte activation, namely the increase in intracellular Ca2+ concentration and the increase in lymphocyte plasma membrane permeabilities to monovalent cations.     

Potassium channels from chick lens epithelium

The technique of patch-voltage clamp has been used to demonstrate several different kinds of K+ channels in the apical membrane of the chick lens epithelium. These include a 200- to 250-ps Ca2+-activated channel, a 200- to 250-ps non-Ca2+-activated channel whose probability of being open increases with hyperpolarization, a 35-ps flickery channel, and a 30-ps inward rectifier, all characterized in symmetrical 150 mM K+. The inward rectifier allows little if any outward current. The probability that the channel is open increases as the membrane patch is depolarized, whereas the mean open and closed times of the channel decrease with depolarization. It is proposed that the rectification is a property of the open channel rather than of its gating. External Cs+ at micromolar concentrations produces a flickery block that increases with hyperpolarization and blocker concentration. The mean open time inside bursts decreases with increasing blocker concentration, whereas the mean intraburst closed time is unaffected. Thus, Cs+ blocks the open channel. The steepness of the voltage dependence of the block suggests that multiple occupancy of the channel is possible.