Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Calcium-activated cation channel in rat thyroid follicular cells

Using the patch-clamp single-channel current recording technique, a cation channel in the contraluminal membrane of rat thyroid follicular cells has been characterized. The channel has a unit conductance of about 35 pS and is equally permeable to sodium and potassium. The pattern of channel opening and closing is independent of the membrane potential. The channel is only operational when the ionized calcium concentration in the fluid which is in contact with the inside of the membrane is at least 1 microM. This conductance pathway can be classified as a calcium dependent non-selective cation channel and could explain stimulant-evoked depolarizations in the thyroid follicular cells.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.

The role of ion channels in the initial steps following exposure of SF-9 lepidopteran insect cells in culture to the delta-endotoxin CryIC from the insecticidal bacterium Bacillus thuringiensis was investigated using single ionic channel measurements and microspectrofluorescence of the calcium-sensitive probe fura-2. It was found that: (1) the toxin triggers an immediate rise in intracellular calcium; (2) the surge is due to calcium entering the cells via calcium channels; (3) the toxin recruits or introduces anionic channels in the cell's plasma membrane in a time-dependent manner. These channels, not seen in the absence of the toxin, are induced by toxin exposure to either side of the cell membrane. They have a conductance of 26 picosiemens (pS) and are mainly permeable to chloride. This study provides the first evidence of the primary role of calcium and chloride ions in the action of delta-endotoxin on cultured insect cells.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris+, N(C2H5)4+, Hepes-) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.     

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.     

Polycystin-2 is an intracellular calcium release channel

Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.     

Hg(2+) affects the intracellular free Ca(2+) oscillatory pattern and the correlated membrane conductance changes in Mg(2+)-stimulated oocytes of the prawn Palaemon serratus

The impact of mercuric ions (Hg(2+)) on prawn oocytes was examined. Prawn oocytes constitute an unusual system in that they are activated at spawning by seawater Mg(2+), which mediates correlated dynamic changes in intracellular free calcium concentration [(Ca(2+))(i)] and membrane conductance associated with the meiosis resumption. Using a voltage clamp technique and intracellular calcium measurements, we observed that treatment with Hg(2+) (5, 10, and 20 microM) resulted in simultaneous impairments of both (Ca(2+))(i) and membrane current responses to external Mg(2+). Treatment with Hg(2+) also resulted in a gradual dose-dependent slow increase in the baseline level of both (Ca(2+))(i) and membrane conductance, independent of stimulation with external Mg(2+). The effect of Hg(2+) on (Ca(2+))(i) and membrane conductance changes resulted from a block of the signal transduction pathway at some point before the InsP(3) receptor channel involved in Ca(2+) release from the endoplasmic reticulum (ER) stocks. The Hg(2+)-dependent gradual increase in both (Ca(2+))(i) and membrane conductance baseline levels may potentially result from a slow permeabilization of the ER membrane, resulting in Ca(2+) leaking into the cytosol. Indeed, this effect could be blocked with the cell permeable Hg(2+) competitor dithiothreitol, which was able to displace Hg(2+) from its intracellular target regardless of whether external Ca(2+) was present or not.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris⁺, N(C₂H₅)₄⁺, Hepes^?) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca~(2+) ([Ca~(2+)]_(cyt)) is an ubiquitous second messenger in plant cell signaling, and [Ca~(2+)]_(cyt) elevation is associated with Ca~(2+)-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca~(2+) channels and their regulation remains limited in planta. A type of voltage-dependent Ca~(2+)-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba~(2+) and Ca~(2+), and their activities can be inhibited by micromolar Gd~(3+). The unitary conductance and the reversal potential of the channels depend on the Ca~(2+) or Ba~(2+) gradients across the plasma membrane. The inward whole-cell Ca~(2+) (Ba~(2+)) current, as well as the unitary current amplitude and NP. of the single Ca~(2+) channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NP_o of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Urinary proteases degrade epithelial sodium channels

Summary The mammalian urinary bladder epithelium accommodates volume changes by the insertion and withdrawal of cytoplasmic vesicles. Both apical membrane (which is entirely composed of fused vesicles) and the cytoplasmic vesicles contain three types of ionic conductances, one amiloride sensitive, an-other a cation-selective conductance and the third a cation conductance which seems to partition between the apical membrane and the mucosal solution. The transport properties of the apical membrane (which has been exposed to urine in vivo) differ from the cytoplasmic vesicles by possessing a lower density of amiloride-sensitive channels and a variable level of leak conductance. It was previously shown that glandular kallikrein was able to hydrolyze epithelial sodium channels into the leak conductance and that this leak conductance was further degraded into a channel which partitioned between the apical membrane and the mucosal solution. This report investigates whether kallikrein is the only urinary constituent capable of altering the apical membrane ionic permeability or whether other proteases or ionic conditions also irreversible modify apical membrane permeability.Alterations of mucosal pH, urea concentrations, calcium concentrations or osmolarity did not irreversible affect the apical membrane ionic conductances. However, urokinase and plasmin (both serine proteases found in mammalian urine) were found to cause an irreversible loss of amiloride-sensitive current, a variable change in the leak current as well as the appearance of a third conductance which was unstable in the apical membrane and appears to partition between the apical membrane and the mucosal solution. Amiloride protects the amiloride-sensitive conductance from hydrolysis but does not protect the leak pathway. Neither channel is protected by sodium. Fluctuation analysis demonstrated that the loss of amiloride-sensitive current was due to a decrease in the sodium-channel density and not a change in the single-channel current. Assuming a simple model of sequential degradation, estimates of single-channel currents and conductances for both the leak channel and unstable leak channel are determined.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes.

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ greater than Cs+ greater than Na+ greater than K+ greater than Li+. The conductance of the membrane was increased up to a value of about 10-2 ohm-1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn+ greater than Cd2+ greater than Ca2+ greater than Sr2+ greater than Mg2+.     

A class of non-selective cation channels in human fibroblasts

Non-selective cation channels were detected in membrane patches of cultured human fibroblasts. The channels had a unitary conductance which ranged from 14 to 25 pS in symmetrical 130 mM NaCl and were permeable to both sodium and potassium ions. Open channel probability was dependent either on the membrane potential and the Ca2+ concentration on the intracellular side of the membrane. High Ca2+ concentrations in the millimolar range were needed to keep the channel active.     

Cloning of a stretch-inhibitable nonselective cation channel

A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane.     

Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes.

Protein K, a recently described outer membrane protein correlated with encapsulation in Escherichia coli (Paakkanen et al., J. Bacteriol. 139:835-841, 1979), has been purified to apparent homogeneity. Purification was based upon the noncovalent association of protein K with peptidoglycan, and the purified protein was shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. Incorporation of small amounts (10(-10) to 10(-11) M) of purified protein K into artificial lipid bilayers resulted in an increase, by many orders of magnitude, in membrane conductance. The increased conductance resulted from the formation of large, water-filled, ion-permeable channels exhibiting single-channel conductance in 1.0 M KCl of 1.83 nS. The membrane conductance showed a linear relationship between current and applied voltage and was not voltage induced or regulated. The channel was permeable to large organic ions (e.g., Tris+ Cl-) and, based upon a pore length of 7.5 nm, a minimum channel diameter of 1.2 nm was estimated; these properties resemble values for other enteric porins. The possible biological role of the pores produced by protein K is discussed.     

爪蟾胚胎脊髓胆碱能神经元上两类非去极化激...

Single calcium channel current was studied on the identified spinal cholinergic neurons from embryonic Xenopus laevis with patch clamp method. The results indicate that some calcium channels show opening activity at resting membrane potential. According to the characteristics of conductance and kinetics of such channels, they could be divided into two types: a stretch sensitive Type-NS with a slope conductance of 7.5 pS (mean open time 0.58 ms at resting membrane potential) and a Type-NL with a slope conductance of 16.7 pS (and two opening times of 2 ms and 19.3 ms). Both types of channel are predominantly active at resting potential and negative membrane phases. It is suggested that they may be involved in the calcium-dependent neuronal events at resting state.     

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.     

Basolateral membrane K permselectivity and regulation in bullfrog cornea epithelium

Summary Ion channels permeable to barium and calcium were reconstituted from theAplysia nervous system into phospholipid bilayers formed on the tips of patch electrodes. With asymmetrical concentrations of barium or calcium on the two sides of the bilayer, the single-channel currents reversed at the calculated barium or calcium reversal potentials, indicating that the channels were cation selective. Channels with conductances of 10, 25 and 36 pS were routinely observed. Calcium and barium were equally effective as charge carriers for the 36-pS channel, whereas magnesium was at least fifteenfold less effective. The gating of all three channels was independent of the voltage across the bilayer, but was affected by the dihydropyridine calcium channel agonist Bay K 8644 (Bay K). In the presence of Bay K but not in its absence, long discrete gating events were routinely observed, suggesting that the dihydropyridine increased the probability of long open states as it does for calcium channels in other systems.Bilayers invariably contained more than a single channel (or conductance state). This was observed even when theAplysia nervous system membranes were prepared in the presence of cytoskeleton disrupting agents, or when the membrane proteins were diluted extensively with exogenous phospholipid. Furthermore, transitions between conductance levels were observed with high frequency. These findings, together with the fact that all of the conductance states share certain properties including voltage-independence and sensitivity to Bay K, suggest that the apparent multiple channel types may in fact represent subconductance states of a single ion channel.     

爪蟾胚胎脊髓胆碱能神经元上两类非去极化激活的钙通道

用膜片箝方法,在标定的爪蟾胚胎脊髓胆碱能神经细胞膜上记录钙离子单通道电流。结果显示,在静息膜电位时钙通道即有开放活动。根据通道的电导及动力学等特性,我们将其分为两种类型:NS 型钙通道,斜率电导7.5pS,静息膜电位时平均开放时间0.58ms,其活动受牵张膜片的影响;NL 型钙通道,斜率电导16.7pS,静息膜电位时二级平均开放时间分别是2ms 和19.3ms。鉴于它们在膜静息以及负电位相的显著活动,我们推测这两类钙通道可能参与神经元静息膜电位时钙依赖的细胞活动过程。     

BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K(+) conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca(2+) increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca(2+)-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP(3)R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-beta-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K(+) conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca(2+) in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP(3)Rs negated the effect of methyl-beta-cyclodextrin, providing further support that IP(3)Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca(2+)-sensitive channels to their second messenger source.