大鼠胰腺β细胞离子通道的一些特性

实验以单个Ｗｉｓｔａｒ大鼠胰腺β细胞为对象,用穿孔膜片箝和细胞贴附式记录技术研究ＡＴＰ敏感Ｋ＾＋通道（ＫＡＴＰ）、延迟整流型Ｋ＾＋通道（ＫＤＲ）、Ｃａ＾２＋通道和Ｎａ＾＋通道的有关特性。结果表明：⑴ＫＡＴＰ通道的内流电导约６５ｐＳ,外流电导约３１ｐＳ,反转电位在－６０ｍＶ左右;⑵ＫＤＲ通道在延迟２０ｍｓ后达到最大激活,ＫＤＲ电流约为ＫＡＴＰ的１／３;⑶钙电流在０ｍＶ左右达到４０－６０ｐＡ的峰值,Ｌ     

Development of ionic channels during mouse neuronal differentiation

Using a mouse embryonal teratocarcinoma (E.C.) cell line, it was possible to follow the sequence of development of ionic channels during neuronal differentiation, with patch-clamp techniques. 1003 E.C. cells were induced to differentiate into neurons by culturing them in defined medium without foetal calf serum (DARMON et al., 1981). Non-differentiated cells were not excitable and presented mainly 2 types of K+ channels: a Ca2+ activated K+ channel (220 pS in symmetrical K+) and a delayed rectifier (30 pS in symmetrical K+). When the cells start to grow neurites, a low threshold calcium current can be recorded, only if the cell is held at hyperpolarized potentials (-70 to -80 mV). Fully differentiated cells with long neurites presented a complete repertoire of ionic channels: voltage dependent Na+ and Ca2+ channels, Ca2+ activated K+ channel and K+ delayed rectifier.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from - 100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.     

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.     

Ion channels in opossum kidney cells.

This review discusses the activation of ion transport pathways during regulatory volume decrease in opossum kidney (OK) cells. OK cells regulate their volume when exposed to a hypotonic medium. The changes in cell volume are caused by activation of ion transport pathways and the accompanying osmotically driven water movement so that the increased cell volume returns toward physiological levels. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease. In OK cells separate K+ and Cl- conductances are activated. The Na+/H+ cotransport system seems not to be involved. The potassium pathway is mediated by a K+ channel with a slope conductance of about 12 pS. The occasionally observed widely distributed Ca2(+)- and voltage-dependent K+ channel of large unit conductance (120 pS) seems not to be involved. The volume regulatory decrease is accompanied by a cell depolarization from a resting potential of about -60 mV to about -20 mV followed by a repolarization. It will be discussed whether the depolarization is caused by the observed activation of stretch-sensitive ion channels of about 30 and 40 pS, respectively. The transient behavior of the cell volume parallels the time-dependent change of the total membrane current. For both recording techniques the volume regulatory decrease can be blocked by quinine. In addition an inward rectifying K+ channel of about 80 pS has been observed in high KCl solution.     

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.     

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.     

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.     

Changes in densities and kinetics of delayed rectifier potassium channels during neuronal differentiation

Single-channel K+ currents were recorded from young and mature spinal neurons cultured from Xenopus embryos to examine the bases of the developmental increases in density and in rate of activation of the macroscopic voltage-dependent delayed rectifier K+ current (IKv). K+ channels of three conductance classes (integral of 80, 30, and 15 pS) are present at both ages, but only the intermediate and small conductance classes are voltage-dependent and thus underlie IKv. The increase in the density of IKv is due to increases in the numbers of intermediate and small channels per cell, but not to changes in their open probabilities. The increase in rate of activation of IKv results from a change in the activation kinetics of the intermediate channel class alone.     

K+ channel cAMP activated in guinea pig gallbladder epithelial cells

In guinea pig gallbladder epithelial cells, an increase in intracellular cAMP levels elicits the rise of anion channel activity. We investigated by patch-clamp techniques whether K(+) channels were also activated. In a cell-attached configuration and in the presence of theophylline and forskolin or 8-Br-cAMP in the cellular incubation bath, an increase of the open probability (P(o)) values for Ca(2+)-activated K(+) channels with a single-channel conductance of about 160 pS, for inward current, was observed. The increase in P(o) of these channels was also seen in an inside-out configuration and in the presence of PKA, ATP, and cAMP, but not with cAMP alone; phosphorylation did not influence single-channel conductance. In the inside-out configuration, the opioid loperamide (10(-5) M) was able to reduce P(o) when it was present either in the microelectrode filling solution or on the cytoplasmic side. Detection in the epithelial cells by RT-PCR of the mRNA corresponding to the alpha subunit of large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) indicates that this gallbladder channel could belong to the BK family. Immunohistochemistry experiments confirm that these cells express the BK alpha subunit, which is located on the apical membrane. Other K(+) channels with lower conductance (40 pS) were not activated either by 8-Br-cAMP (cell-attached) or by PKA + ATP + cAMP (inside-out). These channels were insensitive to TEA(+) and loperamide. The data demonstrate that under conditions that induce secretion, phosphorylation activates anion channels as well as Ca(2+)-dependent, loperamide-sensitive K(+) channels present on the apical membrane.     

A patch-clamp study of histamine-secreting cells

The ionic conductances in rat basophilic leukemia cells (RBL-2H3) and rat peritoneal mast cells were investigated using the patch-clamp technique. These two cell types were found to have different electrophysiological properties in the resting state. The only significant conductance of RBL-2H3 cells was a K+-selective inward rectifier. The single channel conductance at room temperature increased from 2-3 pS at 2.8 mM external K+ to 26 pS at 130 mM K+. This conductance, which appeared to determine the resting potential, could be blocked by Na+ and Ba2+ in a voltage-dependent manner. Rat peritoneal mast cells had a whole-cell conductance of only 10-30 pS, and the resting potential was close to zero. Sometimes discrete openings of channels were observed in the whole-cell configuration. When the Ca2+ concentration on the cytoplasmic side of the membrane was elevated, two types of channels with poor ion specificity appeared. A cation channel, observed at a Ca2+ concentration of approximately 1 microM, had a unit conductance of 30 pS. The other channel, activated at several hundred micromolar Ca2+, was anion selective and had a unit conductance of approximately 380 pS in normal Ringer solution and a bell-shaped voltage dependence. Antigenic stimulation did not cause significant changes in the ionic conductances in either cell type, which suggests that these cells use a mechanism different from ionic currents in stimulus-secretion coupling.     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.     

Expression and modulation of the intermediate- conductance Ca2+-activated K+ channel in glioblastoma GL-15 cells.

We report here the expression and properties of the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channel in the GL-15 human glioblastoma cell line. Macroscopic IK(Ca) currents on GL-15 cells displayed a mean amplitude of 7.2+/-0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC(50)=257 nM), TRAM-34 (IC(50)=55 nM), and charybdotoxin (CTX, IC(50)=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK(Ca) channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K(D) for Ca(2+) of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 muM, for 5 days) virtually suppressed the IK(Ca) current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17+/-0.75 pA/pF at 1-2 days, and 3.11+/-1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IK(Ca) current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IK(Ca) channel mRNA resulting from ERK inhibition and a approximately 50% reduction with time in culture.     

Ionic channels of the vacuolar membrane of higher plants

The vacuole is the major storing compartment of the plant cell. There are two different H(+)-translocating pumps generating transmembrane pH gradients and several types of calcium, anion and potassium channels. The fast-activating vacuolar (FV) current dominates at the physiological level of cytoplasmic Ca2+. An increase in the cytoplasmic Ca2+ concentration causes a decrease in the activity of FV channels and a shift of the membrane potential to more negative values. In this case voltage-dependent Ca2+ channels capable of mediating the vacuolar Ca2+ release can be activated. Moreover, an increase in the cytoplasmic Ca2+ concentration activated vacuolar K+ channels described in the vacuolar membrane of stomatal guard cells. A role of slow-activating channels in the Ca(2+)-induced Ca2+ release is rather improbable.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.