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.     

Properties of a native cation channel activated by Ca2+ store depletion in vascular smooth muscle cells

Depletion of intracellular Ca(2+) stores activates capacitative Ca(2+) influx in smooth muscle cells, but the native store-operated channels that mediate such influx remain unidentified. Recently we demonstrated that calcium influx factor produced by yeast and human platelets with depleted Ca(2+) stores activates small conductance cation channels in excised membrane patches from vascular smooth muscle cells (SMC). Here we characterize these channels in intact cells and present evidence that they belong to the class of store-operated channels, which are activated upon passive depletion of Ca(2+) stores. Application of thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca(2+) ATPase, to individual SMC activated single 3-pS cation channels in cell-attached membrane patches. Channels remained active when inside-out membrane patches were excised from the cells. Excision of membrane patches from resting SMC did not by itself activate the channels. Loading SMC with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), which slowly depletes Ca(2+) stores without a rise in intracellular Ca(2+), activated the same 3-pS channels in cell-attached membrane patches as well as whole cell nonselective cation currents in SMC. TG- and BAPTA-activated 3-pS channels were cation-selective but poorly discriminated among Ca(2+), Sr(2+), Ba(2+), Na(+), K(+), and Cs(+). Open channel probability did not change at negative membrane potentials but increased significantly at high positive potentials. Activation of 3-pS channels did not depend on intracellular Ca(2+) concentration. Neither TG nor a variety of second messengers (including Ca(2+), InsP3, InsP4, GTPgammaS, cyclic AMP, cyclic GMP, ATP, and ADP) activated 3-pS channels in inside-out membrane patches. Thus, 3-pS nonselective cation channels are present and activated by TG or BAPTA-induced depletion of intracellular Ca(2+) stores in intact SMC. These native store-operated cation channels can account for capacitative Ca(2+) influx in SMC and can play an important role in regulation of vascular tone.     

Ca(2+) -independent effects of BAPTA and EGTA on single-channel Cl(-) currents in brown adipocytes

The Cl(-) channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl(-) channels (ORCC). To study tentative Ca(2+) regulation of these channels, we attempted to control Ca(2+) levels at the cytoplasmic side of the inside-out membrane patches with Ca(2+)-chelating agents. However, we found that the commonly used Ca(2+)-chelators EGTA and BAPTA by themselves influenced the Cl(-) channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca(2+)-chelators (acting from the cytoplasmic side) on the single Cl(-) channel currents in patch-clamp experiments. Using fixed (1-2 mM) concentrations of chelators, two types of Cl(-) channels were identified, as discriminated by their reaction to the Ca(2+)-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca(2+). The anionic classical Cl(-) channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca(2+) level control in themselves could fundamentally affect the behaviour of Cl(-) channels.     

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.     

The store-operated calcium entry pathways in human carcinoma A431 cells: functional properties and activation mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca2+ from intracellular Ca2+ stores and activation of Ca2+ influx across the plasma membrane. Two types of Ca2+ channels, highly Ca2+-selective ICRAC and moderately Ca2+-selective ISOC, support store-operated Ca2+ entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated Imin/ICRACL plasma membrane Ca2+ influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca2+ influx pathways in A431 cells. We discovered that (a) ICRAC and ISOC are present in A431 cells; (b) ICRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca2+ store depletion; (c) ISOC currents are moderately selective for divalent cations (PBa/PCs = 14.5) and require at least 300 s for full activation; (d) ICRAC and ISOC currents are activated by PLC-coupled receptor agonists; (e) ISOC currents are supported by Imin/ICRACL channels that display 8.5-10 pS conductance for sodium; (f) ICRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) Imin/ICRACL channels are activated in excised patches by an amino-terminal fragment of InsP3R1 (InsP3R1N); and (h) InsP3 binding to InsP3R1N is necessary for activation of Imin/ICRACL channels. Our findings provide novel information about store-operated Ca2+ influx pathways in A431 cells.     

A store-operated calcium channel in Drosophila S2 cells

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca(2+)-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca(2+)-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20-50 pA at -110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing approximately 300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ > Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of approximately 50 nM, and was also blocked by 20 microM SKF 96365 and by 20 microM 2-APB. At concentrations between 5 and 14 microM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.     

Single-channel recordings of recombinant inositol trisphosphate receptors in mammalian nuclear envelope.

Inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) are intracellular Ca(2+) channels gated by the second messenger InsP(3). Here we describe a novel approach for recording single-channel currents through recombinant InsP(3)Rs in mammalian cells that applies patch-clamp electrophysiology to nuclei isolated from COS-7 cells transiently transfected with the neuronal (SII(+)) and peripheral (SII(-)) alternatively-spliced variants of the rat type 1 InsP(3)R. Single channels that were activated by InsP(3) and inhibited by heparin were observed in 45% of patches from nuclei prepared from transfected cells overexpressing recombinant InsP(3)Rs. In contrast, nuclei from cells transfected with the vector alone had InsP(3)-dependent channel activity in only 1.5% of patches. With K(+) (140 mM) as the permeant ion, recombinant SII(+) and SII(-) channels had slope conductances of 370 pS and 390 pS, respectively. The recombinant channels were 4-fold more selective for Ca(2+) over K(+), and their open probabilities were biphasically regulated by cytoplasmic [Ca(2+)]. This approach provides a powerful new methodology to study the permeation and gating properties of recombinant mammalian InsP(3)Rs in a native mammalian membrane environment at the single-channel level.     

Ca2+ depletion and inositol 1,4,5-trisphosphate-evoked activation of Ca2+ entry in single guinea pig hepatocytes

Store-operated Ca(2+) entry was investigated by monitoring the Ca(2+)-dependent K(+) permeability in voltage-clamped guinea pig hepatocytes. In physiological conditions, intracellular Ca(2+) stores are discharged following agonist stimulation, but depletion of this stores can be achieved using Ca(2+)-Mg(2+)-ATPase inhibitors such as 2,5-di(tert-butyl)-1,4-benzohydroquinone and thapsigargin. The effect of internal Ca(2+) store depletion on Ca(2+) influx was tested in single cells using inositol 1,4,5-trisphosphate (InsP(3)) release from caged InsP(3) after treatment of the cells with 2, 5-di(tert-butyl)-1,4-benzohydroquinone or thapsigargin in Ca(2+)-free solutions. We show that the photolytic release of 1-d-myo-inositol 1,4-bisphosphate 5-phosphorothioate, a stable analog of InsP(3), and Ca(2+) store depletion have additive effects to activate a high level of Ca(2+) entry in single guinea pig hepatocytes. These results suggest that there is a direct functional interaction between InsP(3) receptors and Ca(2+) channels in the plasma membrane, although the nature of these Ca(2+) channels in hepatocytes is unclear.     

Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.     

Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum.

The cerebellar inositol 1,4,5-trisphosphate (InsP3) receptor is a high molecular weight glycoprotein abundantly expressed in Purkinje cells. The subunit structure of the InsP3 receptor protein was examined by cross-linking experiments. Agarose-polyacrylamide gel electrophoresis of the cross-linked materials demonstrated that the cerebellar InsP3 receptor protein is composed of four noncovalently bound identical subunits each with a Mr of 320,000 in both purified and microsome-bound states. Chromatography of the purified receptor on a calmodulin-Sepharose column demonstrated a Ca2(+)-dependent interaction of the InsP3 receptor with calmodulin. Photoaffinity labeling of the cerebellar microsomal fraction with [alpha-32P]8-azidoadenosine 5'-triphosphate revealed the presence of ATP-binding site in the InsP3 receptor. Scatchard analysis of the purified InsP3 receptor revealed the Bmax and Kd values for ATP binding of 2.3 pmol/micrograms and 17 microM, respectively. Reconstitution of the purified InsP3 receptor into the planar lipid bilayer indicated channel activity in the purified receptor. It exhibited a calcium conductance (26 pS in 53 mM Ca2+) and sodium conductance (21 pS in 100-500 mM asymmetric Na+ solutions) with permeability ratios of PCa/PTris = 6.3 and PNa/PCl = 5.4. The purified channel was activated with submillimolar ATP in the presence of InsP3 and modified to reach a large conductance state.     

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.     

Calcium permeability and modulation of nicotinic acetylcholine receptor-channels in rat parasympathetic neurons.

Neuronal nicotinic acetylcholine (ACh)-activated currents in rat parasympathetic ganglion cells were examined using whole-cell and single-channel patch clamp recording techniques. The whole-cell current-voltage (I-V) relationship exhibited strong inward rectification and a reversal (zero current) potential of -3.9 mV in nearly symmetrical Na+ solutions (external 140 mM Na+/internal 160 mM Na+). Isosmotic replacement of extracellular Na+ with either Ca2+ or Mg2+ yielded the permeability (Px/PNa) sequence Mg2+ (1.1) > Na+ (1.0) > Ca2+ (0.65). Whole-cell ACh-induced current amplitude decreased as [Ca2+]0 was raised from 2.5 mM to 20 mM, and remained constant at higher [Ca2+]0. Unitary ACh-activated currents recorded in excised outside-out patches had conductances ranging from 15-35 pS with at least three distinct conductance levels (33 pS, 26 pS, 19 pS) observed in most patches. The neuronal nicotinic ACh receptor-channel had a slope conductance of 30 pS in Na+ external solution, which decreased to 20 pS in isotonic Ca2+ and was unchanged by isosmotic replacement of Na+ with Mg2+. ACh-activated single channel currents had an apparent mean open time (tau 0) of 1.15 +/- 0.16 ms and a mean burst length (tau b) of 6.83 +/- 1.76 ms at -60 mV in Na+ external solution. Ca(2+)-free external solutions, or raising [Ca2+]0 to 50-100 mM decreased both the tau 0 and tau b of the nAChR channel. Varying [Ca2+]0 produced a marked decrease in NP0, while substitution of Mg2+ for Na+ increased NP0. These data suggest that activation of the neuronal nAChR channel permits a substantial Ca2+ influx which may modulate Ca(2+)-dependent ion channels and second messenger pathways to affect neuronal excitability in parasympathetic ganglia.     

Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)- gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single- channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single- ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.     

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.     

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes

K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Voltage-dependent calcium entry in confluent bovine capillary endothelial cells.

Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.     

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.     

Regulatory and spatial aspects of inositol trisphosphate-mediated calcium signals

Hormones that act to release Ca2+ from intracellular stores initiate a signaling cascade that culminates in the production of inositol 1,4,5-trisphosphate (InsP3). The Ca2+ response mediated by InsP3 is not a sustained increase in the cytosolic Ca2+ concentration, but rather a series of periodic spikes that manifest as waves in larger cells. In vitro studies have determined that the key positive feedback parameter driving spikes and waves is a highly localized direct Ca(2+)-activation of InsP3-gated Ca2+ channels. Advances in fluorescent Ca2+ imaging have facilitated the resolution of individual positive feedback units. These studies have revealed that there are several modes of channel coupling underlying global Ca2+ signals; single channel openings or Ca2+ "blips," synchronized clusters of channels or Ca2+ "puffs," and cell wide calcium waves. It appears that the channel clusters that produce Ca2+ puffs are synchronized by the highly localized positive feedback that was predicted by the in vitro studies of channel regulation. Localization of InsP3-induced Ca2+ signals has been shown to be important for activation of several cellular processes including uni-directional salt flow and mitochondrial activation.     

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.