Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings.

The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.     

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.     

Antibody activates cationic channels via second messenger Ca2+

Patch-clamp recordings were used to study single channels permeable to multiple cations in a macrophage cell line. At least three conductance levels were found, consistent with the existence of several types of nonselective cation channels or a single channel with multiple open states. The activity of the channels depended very little on voltage but was affected by internal Ca2+ concentration. Specific subclasses of immunoglobulins (IgG1 and IgG2b) bound to an Fc receptor on the surface of these macrophages. When an IgG2b was applied to the cell exterior after a patch pipette had been sealed in the cell-attached mode, the nonselective cation channels within the patch were activated. Thus, these channels must be modulated by a second messenger. Since antibodies binding to the Fc receptor have been shown to produce a rise in intracellular Ca2+, this cation must be considered a candidate as a second messenger that amplifies the effect of antibody in gating these channels.     

Signal transduction and ion channels in guard cells.

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity. At the plasmalemma, loss of K+ requires depolarization of the membrane potential into the range at which the outward K+ channel is open. ABA-induced activation of a non-specific cation channel, permeable to Ca2+, may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are activated by Ca2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K+ current at any given membrane potential; this activation is Ca(2+)-independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C. Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca(2+)-activated, have been identified which are capable of K+ efflux; these are the voltage-independent VK channel specific to K+, and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K+ and Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN     

The role of calcium ions in the process of acetyltransferase activation during the formation of platelet-activating factor (PAF-acether).

The role of Ca2+ in the activation of the enzyme lyso-(platelet-activating factor): acetyl-CoA acetyltransferase was studied in rat peritoneal macrophages in response to complement-coated zymosan particles and ionophore A23187. By using Ca2+-containing buffers, a threshold concentration of extracellular Ca2+ above 1 microM was found to be necessary to observe the activation of the enzyme in response to zymosan. By contrast, a significant role of intracellular Ca2+ in this process could be ruled out, since the putative intracellular calcium-transport antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] did not inhibit the activation of the acetyltransferase induced by zymosan in the presence of extracellular Ca+. The link between acetyltransferase activation and extracellular Ca2+ transport was studied by measuring Ca2+ uptake in response to the stimuli. Zymosan particles induced a rapid increment in cell-associated Ca2+ which correlated well with the extent of acetyltransferase activation (r = 0.91) and with the release of platelet-activating factor (r = 0.95) in response to different doses of zymosan. Cellular Ca2+ efflux in response to zymosan particles was also measured and found to be increased, as compared with controls, when the activation of the acetyltransferase declined. In short, the data suggest that the entry of extracellular Ca2+ into the cell is a crucial event in the activation of acetyltransferase and, thereby, in the formation of platelet-activating factor in rat peritoneal macrophages.     

Calcium channels activated by depletion of internal calcium stores in A431 cells.

Depletion of intracellular calcium stores induces transmembrane Ca2+ influx. We studied Ca(2+)- and Ba(2+)-permeable ion channels in A431 cells after store depletion by dialysis of the cytosol with 10 mM BAPTA solution. Cell-attached patches of cells held at low (0.5 microM) external Ca2+ exhibited transient channel activity, lasting for 1-2 min. The channel had a slope conductance of 2 pS with 200 mM CaCl2 and 16 pS with 160 mM BaCl2 in the pipette. Channel activity quickly ran down in excised inside-out patches and was not restored by InsP3 and/or InsP4. Thapsigargin induced activation in cells kept in 1 mM external Ca2+ after BAPTA dialysis. These channels represent one Ca2+ entry pathway activated by depletion of internal calcium stores and are clearly distinct from previously identified calcium repletion currents.     

Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl- currents (I(ClCa)) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, I(ClCa) was evoked immediately upon membrane rupture but then exhibited marked rundown to approximately 20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5'-(beta,gamma-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, I(ClCa) was approximately 100% of initial levels. I(ClCa) recorded with AMP-PNP-containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in I(ClCa) was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater "on" rates, and voltage-dependent closing steps ("off" rates). Our model reproduced well the Ca2+ and voltage dependence of I(ClCa) as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl- channel complex influences current generation dramatically through one or more critical voltage-dependent steps.     

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.     

Store-operated cationic channels in human myeloid leukaemia K562 cells

Using the whole-cell patch clamp technique, single channels operated by intracellular Ca(2+)-store depletion were first revealed in human myeloid leukaemia cells K562. A single store-operated channel could be detected in divalent-free extracellular solutions with Na+ as a permeant ion, and intracellular solutions with strong Ca(2+)-helating agent with some delay after whole-cell formation. Addition of inositol-1,4,5-triphosphate to the pipette solution resulted in a significant decrease of this latency. These channels had a conductance of 29 pS, and were inhibited by low concentration of external Ca2+. Our results enable us to assume that the revealed channels are calcium release-activated calcium channels, operated by Ca2+ depletion of endoplasmic reticulum.     

Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells

The effects of glucose, diazoxide, K+, and tolbutamide on the activity of K+ channels, membrane potential, and cytoplasmic free Ca2+ concentration were investigated in beta-cells from the Uppsala colony of obese hyperglycemic mice. With [K+]e = [K+]i = 146 mM, it was demonstrated that the dominating channel at the resting potential is a K+ channel with a single-channel conductance of about 65 picosiemens and a reversal potential of about +70 mV (pipette potential). This channel is characterized by complex kinetics with openings grouped in bursts. The channel was completely inhibited by 20 mM glucose in intact cells or by intracellularly applied Mg-ATP (1 mM). The number of active channels was markedly reduced already by 5 mM glucose. However, the single channel current of the channels remaining active was unaffected, indicating no major depolarization. To evoke a substantial depolarization of the membrane and thereby action potentials, a total block in channel activity was necessary. This could be achieved either by increasing the concentration of glucose to 20 mM or by combining 5 mM glucose with 100 microM tolbutamide. In both cases, the effect was counteracted by the hyperglycemic sulfonamide diazoxide. The effects on single channel activity were paralleled by changes in membrane potential and cytoplasmic free Ca2+ concentration, also when the latter measurements were performed at room temperature. The transient increase in the number of active channels and the resulting hyperpolarization observed after raising the glucose concentration to 20 mM probably reflected a drop in cytoplasmic ATP concentration. It is suggested that ATP works as a key regulator of the beta-cell membrane potential and thereby the opening of voltage-activated Ca2+ channels.     

Single-channel recordings of two types of calcium channels in rat pancreatic beta-cells.

Using the cell-attached configuration of the patch clamp technique, we have identified two different types of Ca channels in rat pancreatic beta-cell membranes. The two channels differ in single channel conductance, voltage dependence, and inactivation properties. The single-channel conductance, measured with 100 mM Ba2+ in the pipette, was 21.8 pS for the large channel and 6.4 pS for the small channel. The large-conductance channel is similar to the fast deactivating or L-type Ca channel described in other preparations. It is voltage dependent, has a threshold for activation around -30 mV, and can be activated from a holding potential of -40 mV. On the other hand, the small-conductance Ca channel is similar to the SD or T type Ca channel; it has a lower activation threshold, around -50 mV, and it can be inactivated by holding the membrane potential at -40 mV.     

Calcium-activated endonexin II forms calcium channels across acidic phospholipid bilayer membranes

Human endonexin II (annexin V) and recombinant human endonexin II can be activated by Ca2+ to interact with acidic phospholipid bilayers formed at the tip of a patch pipette. Once associated with the bilayer, endonexin II forms voltage-gated channels which are selective for divalent cations according to the following series Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+. However, endonexin II also expresses a selective affinity for Ca2+ which is manifest by an observed reduced current through the open channel when Ca2+ is the charge carrier. La3+ blocks endonexin II channels, as it does synexin (annexin VII) and other types of Ca2+ channels. However, as with synexin, the dihydropyridine Ca2+ channel antagonist nifedipine does not affect endonexin II channel activity. Endonexin II channels are also permeant to Li+, Cs+, Na+, and to a lesser extent, K+, resembling in this manner Ca2+ release channels from sarcoplasmic reticulum. Indeed, the low affinity of endonexin II channels for such ions as Cs+ or Li+ have allowed us to use these cations for measurement of the kinetic properties of the channel, with minimal concerns for the ion/channel interactions observed with the physiological substrate, Ca+. Finally, we observed that endonexin II channel activity always occurred in bursts, making necessary the use of two exponential functions to fit open- and closed-time histograms. We conclude from these data that the domain responsible for endonexin II channel activity, first observed by ourselves in the homologue synexin, is probably the C-terminal tetrad repeat common to both molecules.     

Receptor-activated single channels in intact human platelets.

We report "cell-attached" patch clamp studies of intact human platelets which show receptor-activated single channels. Inclusion of ADP in the patch pipette, but not in the bath, resulted in the appearance of inward currents indicative of single channels tightly coupled to the ADP receptors. The channels had a slope conductance of 11 picosiemens at the resting potential. Removal of 1 mM Ca2+ or replacement of chloride by gluconate in the pipette filling solution had little effect on the slope conductance at the resting potential or on the estimated reversed potential. With isotonic BaCl2 in the pipette, ADP evoked single channel currents with a slope conductance of 10 picosiemens. Thus these channels appear to be permeable to monovalent and divalent cations and selective for cations over anions. Addition of 5 mM Ni2+ (which blocks ADP-evoked rapid calcium entry in fura-2-loaded platelets) to the pipette solution blocked ADP-evoked channel activity. These channels may therefore provide an important mechanism for ADP to activate human platelets within a small fraction of a second.     

Ca2+ and nucleotide dependent regulation of voltage dependent anion channels in the plasma membrane of guard cells.

Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides. Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state. Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s). We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.     

Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats

Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 microM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 microM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+,Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels.     

Patch cramming: monitoring intracellular messengers in intact cells with membrane patches containing detector ion channels.

This paper introduces "patch cramming," a new procedure that utilizes an ion channel gated directly by an intracellular messenger molecule as a probe for detecting changes in the concentration of that molecule in an intact cell. A patch pipette containing the channel in a membrane patch is inserted into a recipient cell where the channel locally "senses" the intracellular messenger. In this study patches containing Ca2(+)-dependent K+ channels were inserted into Helix neurons, where they were activated by Ca2+ influx during trains of action potentials. Channels gated directly by other messengers, including cyclic nucleotides and IP3, have also been identified. Hence, by using detector channels with appropriate specificity, it may be possible to detect local intracellular fluctuations of these molecules.     

Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.     

Receptor-mediated activation of arachidonic acid release in mouse peritoneal macrophages is linked to extracellular calcium influx.

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. Deprivation of external Ca2+ led to strong inhibition of receptor-mediated arachidonic acid release, which was completely restored when Ca2+ was added to the incubation medium. When arachidonic acid release was examined in Ca(2+)-depleted cells, the response took place only in presence of external Ca2+. Verapamil, a voltage-dependent Ca2+ channel blocker, nearly abolished arachidonic acid release in response to both platelet-activating factor and zymosan. These results suggest that extracellular Ca2+ influx is functionally linked to arachidonic acid release and hence to phospholipase A2 activation in mouse peritoneal macrophages.     

Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires beta-subunit

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel alpha(1C)-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel beta(1a)- or beta(2a)-subunit with the alpha(1C)-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in alpha(1C) + beta(1a)-expressing HEK-293 cells was rapid after a depolarization to +100 mV (tau = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5'-O-(2-thiodiphosphate) and guanosine 5'-O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A(1) receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel alpha(1C)-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary beta-subunit and that this facilitation is independent of G protein pathways.     

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.