Phallolysin A mushroom toxin,forms proton and voltage gated membrane channels

Phallolysin, a water soluble protein of M _r 34,000 produced by the poisonous mushroom Amanita phalloides, causes lysis of various mammalian cell types. Lysis is thought to be initiated by the formation of ion permeable membrane channels. We therefore studied the interaction of phallolysin with solvent-free planar lipid bilayers. In the presence of low phallolysin concentrations (10–100 nM) single channel current fluctuations were observed. Unit channel conductances are 44 pS in 500 mM NaCl and 77 pS in 1 M NaCl. Although the channel does not significantly discriminate between alkali cations, its permeability to Cl^- is lower (P _K ⁺/P _Cl ^-=4/1). Gating kinetics display a pronounced bursting behavior and a dependence on membrane voltage, cis side pH-value, and on membrane lipid composition. An equivalence relation between membrane voltage and proton concentration was found, i.e. a pH change of one unit is equivalent to a corresponding voltage change of 130 mV. Dependence on the amount of negatively charged lipids is explained by changes of the actual pH due to surface charge effects.Abbreviations 1,3-SMPC 1-stearoyl-3-myristoyl-glycero-2-phosphocholine - 1,2-DOPS 1,2-dioleoyl-glycero-3-phosphoserine     

Plasma membrane Ca2+ channels in roots of higher plants and their role in aluminium toxicity

Single Ca²⁺ channel records were obtained from plasma membrane-enriched fractions of wheat roots incorporated into artificial planar lipid bilayers. The channel had a unitary conductance of 15 pS for a 10 to 95 mM CaCl₂ gradient (cytoplasm: outside of the cell). The voltage dependence displayed by the channel agreed with that expected for Ca²⁺ channels in the plasma membrane. The channel gating was strongly modified by addition of 20 M extracellular verapamil (a Ca²⁺ channel antagonist). Extracellular AlCl₃ (70 M, pH 4.9) almost completely blocked the channel.     

The characterization of a monovalent cation-selective channel of mammalian cardiac muscle sarcoplasmic reticulum

Summary Rabbit cardiac muscle sarcoplasmic reticulum (SR) was isolated and separated into ryanodine-sensitive and-insensitive fractions (L.R. Jones and S.E. Cala,J. Biol. Chem. 256:11809–11818, 1981). Vesicles of cardiac SR were incorporated into planar phospholipid bilayers by fusion and the channel activity of the membrane studied under voltage-clamp conditions (C. Miller,J. Membrane Biol. 40: 1–23, 1978). Both fractions contain a monovalent cation-selective three-state channel. In the presence of 75mm K₂SO₄, the fully open state () conductance of this channel is 157.2±30 pS and the sub-state () conductance is 100.7±21 pS. Both open states display the same selectivity sequence for monovalent cations, i.e. K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺ and may be blocked by the skeletal muscle relaxants decamethonium and hexamethonium. Block occurs when the compounds are added to either side of the membrane. The properties of the cardiac SR cation channel are compared with those of the previously reported monovalent cation-selective channels of mammalian and amphibian skeletal muscle SR.     

Electrophysiological Properties of Ion Channels in Ascaris suum Tissue Incorporated into Planar Lipid Bilayers

Ion channels are important targets of anthelmintic agents. In this study, we identified 3 types of ion channels in Ascaris suum tissue incorporated into planar lipid bilayers using an electrophysiological technique. The most frequent channel was a large-conductance cation channel (209 pS), which accounted for 64.5% of channels incorporated (n=60). Its open-state probability (P_o) was ~0.3 in the voltage range of −60~+60 mV. A substate was observed at 55% of the main-state. The permeability ratio of Cl⁻ to K⁺ (P_Cl/P_K) was ~0.5 and P_Na/P_K was 0.81 in both states. Another type of cation channel was recorded in 7.5% of channels incorporated (n=7) and discriminated from the large-conductance cation channel by its smaller conductance (55.3 pS). Its Po was low at all voltages tested (~0.1). The third type was an anion channel recorded in 27.9% of channels incorporated (n=26). Its conductance was 39.0 pS and P_Cl/P_K was 8.6±0.8. P_o was ~1.0 at all tested potentials. In summary, we identified 2 types of cation and 1 type of anion channels in Ascaris suum. Gating of these channels did not much vary with voltage and their ionic selectivity is rather low. Their molecular nature, functions, and potentials as anthelmintic drug targets remain to be studied further.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Effects of reagents modifying carboxyl groups on the gating current of the myelinated nerve fiber

Summary K⁺ channels in inside-out patches from hamster insulin tumor (HIT) cells were studied using the patch-clamp technique. HIT cells provide a convenient system for the study of ion channels and insulin secretion. They are easy to culture, form gigaohm seals readily and secrete insulin in response to glucose. The properties of the cells changed with the passage number. For cell passage numbers 48 to 56, five different K⁺-selective channels ranging from 15 to 211 pS in symmetrical 140mm KCl solutions were distinguished. The channels were characterized by the following features: a channel with a conductance (in symmetrical 140mm KCl solutions) of 210 pS that was activated by noncyclic purine nucleotides and closed by H⁺ ions (pH=6.8); a 211 pS channel that was Ca²⁺-activated and voltage dependent; a 185 pS channel that was blocked by TEA but was insensitive to quinine or nucleotides; a 130 pS channel that was activated by membrane hyperpolarization; and a small conductance (15 pS) channel that was not obviously affected by any manipulation. As determined by radioimmunoassay, cells from passage number 56 secreted 917±128 ng/mg cell protein/48 hr of insulin. In contrast, cells from passage number 77 revealed either no channel activity or an occasional nonselective channel, and secreted only 29.4±8.5 ng/mg cell protein/48 hr of insulin. The nonselective channel found in the passage 77 cells had a conductance of 25 pS in symmetrical 140mm KCl solutions. Thus, there appears to be a correlation between the presence of functional K⁺ channels and insulin secretion.     

Ion channels in the plasma membrane ofAmaranthus protoplasts: One cation and one anion channel dominate the conductance

Summary This report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons ofAmaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier.The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K⁺) and relative permeability sequence K⁺>Na⁺>Cl^– (10.160.03); whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of sub-conductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (V _m ) is depolarized; at millimolar levels of [Ca²⁺]_cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2mm) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO ₃ ^– >Cl^–>K⁺>Aspartate (2.0410.18 to 0.090.04). The relative permeability for K⁺ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clampV _m at a preferred value in the face of events which would otherwise perturbV _m .     

Ion-channels reconstituted into lipid bilayer from human sperm plasma membrane

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200 // trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na⁺-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about −18 mV, which is close to the predicated value of a perfect Nernst K⁺ or Na⁺ electrode. In 50//10 mM CaCl₂ solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about −20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction. Mol. Reprod. Dev. 50:354–360, 1998. © 1998 Wiley-Liss, Inc.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

How Does the W434F Mutation Block Current in Shaker Potassium Channels?

The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH₂-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10⁻⁵ or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state.     

Potassium-39 NMR of K+ interaction with the gramicidin channel and NMR-derived conductance ratios for Na+, K+ and Rb+

Summary A potassium-39 NMR study of potassium ion interaction with the gramicidin transmembrane channel in phospholipid bilayers at high ion activity is reported which allows determination of a weak binding constant, K _b ^w 8.3/m, and an off-rate constant for the weak site,k _off ^w 2.6×10⁷/sec. These values are interpreted with the aid of additional NMR data as the binding constant for formation of the doubly occupied channel state and the rate constant for an ion leaving the doubly occupied state. Considering the singly occupied channel state for the potassium ion to be electrically silent at 1 molar ion activity, as with the sodium ion, the single-channel conductance for 100 mV and 30°C calculated to be 29 pS, and using the same approximation with previous NMR results on the sodium and rubidium ions, reasonable conductance ratios were calculated. Further experimental estimates of the other three constants with the experimental location of binding sites and Eyring rate theory to introduce voltage dependence allowed a more complete calculation of the two-site channel. The single-channel conductance for potassium ion is calculated to be 24 pS at 1m activity and 26 pS at 0.6m activity, which compares for diphytanoyl phosphatidylcholine membranes to an experimental most probable single-channel conductance of 25 pS and a mean channel conductance of 20 pS. The calculated conductance ratios using NMR-derived constants were (K)/(Na)=2.0 and (Rb)/(Na)=4.3. These results are close to the experimental values and provide further basis for the use of NMR of quadrupolar ions to provide information on the ionic mechanism of channel transport.     

Patch clamping VDAC in liposomes containing whole mitochondrial membranes

Summary Whole mitochondrial membranes isolated fromNeurospora crassa were reconstituted into liposomes and patch clamped. Clear activity characteristic of the mitochondrial channel VDAC was found, namely: open state conductance of 650 pS (in 150mm KCl, 1mm CaCl₂, 20mm HEPES, pH 7.2), voltage-dependent closure at both positive and negative potentials, change in conductance upon channel closure of about 450 pS in response to negative and positive potentials, and increased voltage dependence in the presence of König's polyanion. This is the first clear demonstration of VDAC single channels using the patch-clamp technique, even though others used this method before to study whole mitochondrial membranes and liposomes containing mitochondrial proteins. We also found one other channel with a conductance change of about 120 pS.     

Chloride channels in the nuclear membrane

Summary Chloride-selective ion channels were measured from isolated rat liver nuclei. Single ion channel currents were recorded in both nuclear-attached and in excised patches in the insideout configuration of the patch-clamp technique. Two types of chloride conductance were defined, a large conductance (150 pS;i _Cl.N ) channel with complex kinetics and multiple substates, and a second smaller conductance (58 pS;I _Cl.n ) channel sensitive to block by ATP. The channels were inhibited by pharmacological agents known to block chloride channels and were insensitive to internal and external changes in calcium and magnesium. Presumably the channels reside in the external membrane of the nuclear double membrane and may mediate charge balance in the release and uptake of calcium from the perinuclear space.     

Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K⁺), and the presence of short and long lasting subconducting states. Its open probability P_o increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca²⁺ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K⁺ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca²⁺] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca²⁺, Ba²⁺ or Mg²⁺-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by µM Ca²⁺ while its Ca²⁺ sensitivity was potentiated by millimolar ATP. However Mg²⁺ and calmodulin modulated its gating behavior. Ca²⁺ releasing drugs such as caffeine and ryanodine increased its P_o. All these features are characteristics of the SR Ca²⁺ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway. This channel displays all the regulatory sites of the native cardiac SR Ca²⁺ release channel. However, when NA was used as charge carrier, multiple subconducting states were observed. In conclusion, the reconstitution experiments have yield a great deal of informations about the biochemical and biophysical events that may regulated the ionic flux across the SR membrane.     

A型流感病毒H5N1的M2离子通道稳定细胞株的建立

A型流感病毒H5N1的M2离子通道(H5M2)基因经优化后由人工合成,适合于哺乳动物细胞中表达.通过酶切克隆于pcDNA4质粒,并在HEK293细胞中建立稳定细胞株.Western blotting和免疫荧光证实H5M2在稳定细胞中只有在四环素诱导下才能表达,并经膜片钳证实在HEK293细胞中表达的H5M2具有H 通道活性,为M2离子通道功能的研究和M2离子通道阻断剂筛选方法的建立提供了参考.     

Functional characterization of P2X3 receptors fused with fluorescent proteins

P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X₃ receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6?pS) and the fast activation kinetics of the receptor (20?ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X₃ constructs carrying a fluorescent tag (ECFP or DsRed₂) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X₃ receptors in CNS signaling.     

TRPC1 protein forms only one type of native store-operated channels in HEK293 cells

TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated I_max channels, while the properties of I_min and I_NS channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current–voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native I_max channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated I_max channels. All these data allow us to conclude that TRPC1 protein forms native store-operated I_max channels but is not an essential subunit for other store-operated channel types in HEK293 cells.     

Permeation properties of inward-rectifier potassium channels and their molecular determinants

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) > Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.     

Characterization of a high-conductance,voltage-dependent cation channel from the plasma membrane of rye roots in planar lipid bilayers

Plasma-membrane vesicles were purified by aqueous-polymer two-phase partitioning of a microsomal membrane fraction from rye (Secale cereale L.) roots and incorporated into planar 1-palmitoyl-2-oleoyl phosphatidylethanolamine bilayers. A high-conductance cation channel (a maxi cation channel) was characterized from single-channel electrical recordings. The channel was incorporated into the bilayer with its cytoplasmic surface facing the trans compartment and voltages were referenced cis with respect to trans. The channel was permeable to both monovalent and divalent cations. The unitary conductance was 451 pS in symmetrical 100 mM KCl and 213 pS in symmetrical 100 mM BaCl₂. The permeability ratio P_KP_Ba was 1.002.56. Unitary conductances declined in the order K⁺Rb⁺>Cs⁺>Na⁺> Li⁺ (monovalent cations) and Ba²⁺>Sr²⁺>Ca²⁺> Mg²⁺>Co²⁺>Mn²⁺ (divalent cations). The relative permeabilities of monovalent cations mirrored their conductivity sequence, whereas the permeabilities of all divalent cations were similar. The maxi cation channel showed complex kinetics, exhibiting both voltage- and time-dependent inactivation and voltage-dependent gating. The voltage dependence of the kinetics shifted in parallel with changes in the reversal potential of the channel. In symmetrical 100 mM KCl, following a voltage step from zero to the test voltage, the channel inactivated and the active-channel lifetime ( _i) shortened exponentially as the test voltage was increased. The channel always opened immediately upon depolarization to zero volts, indicating that inactivation of the channel did not result from the loss of any intrinsic factor. The probability of finding an active channel in the open state (P₀) exhibited a bell-shaped relationship with membrane potential. At voltages between -40 and 80 mV, P₀ exceeded 0.99, but p₀ declined abruptly at more extreme voltages. Under ionic conditions which approximated physiological conditions, in the presence of 100 mM KCl on the trans (cytoplasmic) side and 1 mM KCl plus 2 mM CaCl₂ on the cis (extracellular) side, the reversal potential was 15.6 mV and the kinetics approximated those observed in symmetrical 100 mM KCl. Thus, the channel would open upon depolarization of the plasma membrane in vivo. If the channel functioned physiologically as a Ca²⁺ channel it might be involved in intracellular signalling: the channel could open in response to a variety of environmental, developmental and pathological stimuli which depolarize the plasma membrane, allowing Ca²⁺ into the cytoplasm and thereby initiating a physiological response.Abbreviations E_K Nernst (equilibrium) potential for potassium - E_rev zero-current (reversal) potential - I/V current/voltage - _c apparent mean lifetime of the activated-channel closed state - _i apparent mean lifetime of the activated channel following a voltage step from zero volts - ₀ apparent mean lifetime of the activated-channel open state - PE 1-palmitoyl-2-oleoyl phosphatidylethonlamine - P₀ probability of finding the activated channel in an open state - TEA⁺ tetraethylammonium This work was supported by the Agriculture and Food Research Council and by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Prof. E.A.C. MacRobbie (University of Cambridge, UK).     

Large-Conductance Cation Channels in the Envelope of Nuclei from Rat Cerebral Cortex

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K⁺ is 166 pS. The channels are selective for cations with P _K/P _Na= 2. They are neither permeable to, nor gated by Ca²⁺. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles. Received: 12 November 1996/Revised: 18 February 1997