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1.
The functional role of ligand-gated ion channels depends critically on whether they are predominantly permeable to cations or anions. However, these, and other ion channels, are not perfectly selective, allowing some counterions to also permeate. To address the mechanisms by which such counterion permeation occurs, we measured the anion-cation permeabilities of different alkali cations, Li+ Na+, and Cs+, relative to either Cl− or anions in both a wild-type glycine receptor channel (GlyR) and a mutant GlyR with a wider pore diameter. We hypothesized and showed that counterion permeation in anionic channels correlated inversely with an equivalent or effective hydrated size of the cation relative to the channel pore radius, with larger counterion permeabilities being observed in the wider pore channel. We also showed that the anion component of conductance was independent of the nature of the cation. We suggest that anions and counterion cations can permeate through the pore as neutral ion pairs, to allow the cations to overcome the large energy barriers resulting from the positively charged selectivity filter in small GlyR channels, with the permeability of such ion pairs being dependent on the effective hydrated diameter of the ion pair relative to the pore diameter. 相似文献
2.
The permeation of monovalent organic cations through adenosine 3,5-cyclic monophosphate-(cAMP) activated channels was studied by recording macroscopic currents in excised inside-out membrane patches from the dendritic knobs of isolated mammalian olfactory receptor neurons (ORNs). Current-voltage relations were measured when bathing solution Na+ was replaced by monovalent organic cations. Permeability ratios relative to Na+ ions were calculated from changes in reversal potentials. Some of the small organic cations tested included ammonium (NH
4
+
), hydroxylammonium and formamidinium, with relative permeability ratios of 1.41, 2.3 and 1.01 respectively. The larger methylated and ethylated ammonium ions studied included: DMA (dimethylammonium), TMA (tetramethylammonium) and TEA (tetraethylammonium) and they all had permeability ratios larger than 0.09. Even large cations such as choline, arginine and tris(hydroxymethyl)aminomethane (Tris) were appreciably permeant through the cAMP-activated channel with permeability ratios ranging from 0.19 to 0.7. The size of the permeating cations, as assessed by molecular weight, was a good predictor of the permeability. The permeability sequence of the cAMP-activated channel in our study was PNH4 > PNa > pDMA > pTMA > PCholine > PTEA. Higher permeability ratios of hydroxylammonium, arginine and tris(hydroxymethyl)aminomethane cannot be explained by ionic size alone. Our results indicate that: (i) cAMP-activated channels poorly select between monovalent cations; (ii) the pore dimension must be at least 6.5 × 6.5 Å, in order to allow TEA and Tris to permeate and (iii) molecular sieving must be an important mechanism for the permeation of large organic ions through the channels with specific ion binding playing a smaller role than in other structurally similar channels. In addition, the results clearly indicate that cyclic nucleotide-gated (CNG) channels in different cells are not the same, the olfactory CNG channel being different from that of the photoreceptors, particularly with respect to the permeation of large organic cations, which the ORN channels allow to permeate readily.This work was supported by the Australian Research Council of Australia. 相似文献
3.
Compelling evidence shows that intracellular free magnesium [Mg^2+]i may be a critical regulator of cell activity in eukaryotes. However, membrane transport mechanisms mediating Mg^2+ influx in mammalian cells are poorly understood. Here, we show that mechanosensitive (MS) cationic channels activated by stretch are permeable for Mg^2+ ions at different extracellular concentrations including physiological ones. Single-channel currents were recorded from cell-attached and inside-out patches on K562 leukaemia cells at various concentrations of MgCl2 when Mg^2+ was the only available carrier of inward currents. At 2 mM Mg^2+, inward mechanogated currents representing Mg^2+ influx through MS channels corresponded to the unitary conductance of about 5 pS. At higher Mg^2+ levels, only slight increase of single-channel currents and conductance occurred, implying that Mg^2+ permeation through MS channels is characterized by strong saturation. At 20 and 90 mM Mg^2+, mean conductance values for inward currents carried by Mg^2+ were rather similar, being equal to 6.8 ± 0.5 and 6.4 ± 0.5 pS, respectively. The estimation of the channel-selective permeability according to constant field equation is obviously limited due to saturation effects. We conclude that the detection of single currents is the main evidence for Mg^2+ permeation through membrane channels activated by stretch. Our single-current measurements document Mg^2+ influx through MS channels in the plasma membrane of leukaemia cells. 相似文献
4.
Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions. 相似文献
5.
Block, permeation, and agonist action of small organic amine compounds were studied in acetylcholine receptor (AChR) channels. Single channel conductances were calculated from fluctuation analysis at the frog neuromuscular junction and measured by patch clamp of cultured rat myotubes. The conductance was depressed by a few millimolar external dimethylammonium, arginine, dimethyldiethanolammonium, and Tris. Except with dimethylammonium, the block was intensified with hyperpolarization. A two-barrier Eyring model describes the slowed permeation and voltage dependence well for the three less permeant test cations. The cations were assumed to pause at a site halfway across the electric field of the channel while passing through it. For the voltage-independent action of highly permeant dimethylammonium, a more appropriate model might be a superficial binding site that did not prevent the flow of other ions, but depressed it. Solutions of several amine compounds were found to have agonist activity at millimolar concentrations, inducing brief openings of AChR channels on rat myotubes in the absence of ACh. 相似文献
6.
Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels
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Moorhouse AJ Keramidas A Zaykin A Schofield PR Barry PH 《The Journal of general physiology》2002,119(5):411-425
Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2' Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels. 相似文献
7.
The mitochondrial channel, VDAC, regulates metabolite flux across the outer membrane. The open conformation has a higher conductance and anionic selectivity, whereas closed states prefer cations and exclude metabolites. In this study five mutations were introduced into mouse VDAC2 to neutralize the voltage sensor. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity, and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for wild-type VDAC2. The outer membranes of mitochondria containing the mutant are permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from an anionic to a cationic preference does not substantially influence the flux of negatively charged metabolites. This finding supports our previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall. 相似文献
8.
S S Garber 《Biophysical journal》1988,54(5):767-776
Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions. 相似文献
9.
Beckstead MJ Phelan R Trudell JR Bianchini MJ Mihic SJ 《Journal of neurochemistry》2002,82(6):1343-1351
Strychnine-sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand-gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor alpha1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1-trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand-gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on alpha1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding. 相似文献
10.
We used molecular dynamics (MD) simulations to explore the transport of single cations through the channel of the muscle nicotinic acetylcholine receptor (nAChR). Four MD simulations of 16 ns were performed at physiological and hyperpolarized membrane potentials, with and without restraints of the structure, but all without bound agonist. With the structure unrestrained and a potential of −100 mV, one cation traversed the channel during a transient period of channel hydration; at −200 mV, the channel was continuously hydrated and two cations traversed the channel. With the structure restrained, however, cations did not traverse the channel at either membrane potential, even though the channel was continuously hydrated. The overall results show that cation selective transport through the nAChR channel is governed by electrostatic interactions to achieve charge selectivity, but ion translocation relies on channel hydration, facilitated by a trans-membrane field, coupled with dynamic fluctuations of the channel structure. 相似文献
11.
Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells 总被引:17,自引:1,他引:16
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J Yang 《The Journal of general physiology》1990,96(6):1177-1198
Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth. 相似文献
12.
Functional chloride channels by mammalian cell expression of rat glycine receptor subunit 总被引:15,自引:0,他引:15
H Sontheimer C M Becker D B Pritchett P R Schofield G Grenningloh H Kettenmann H Betz P H Seeburg 《Neuron》1989,2(5):1491-1497
Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency. 相似文献
13.
In this review, we discuss the function and modulation of chloride-selective glycine receptor (GlyR) channels, some genetic
diseases originated from dysfunction of GlyRs, and modulation of glycinergic synapses by intracellular calcium (Ca2+) with particular attention on the motoneurons of the hypoglossal nucleus. This motor nucleus is a brainstem structure implicated
in the command of coordinated movements during oral behavioral phenomena, including feeding, drinking, grooming, and respiration.
In this nucleus, more than 90% of its cells are motoneurons. These hypoglossal motoneurons (HMs) are involved in a variety
of motor functions and exhibit two remarkable features: (i) a low endogenous Ca2+ buffering capacity, which determines the rapid dynamics of cytosolic intracellular Ca2+, and (ii) powerful glycinergic inputs, which determine the main inhibitory drive on the above cells in adult animals. Glycine
receptors belong to the superfamily of Cys-loop ligand-gated ion channels. They are capable of forming functional homo-or
heteromeric chloride-selective channels. Dysfunction of GlyRs results in a genetic neurological motor disorders, including
hyperekplexia. These diseases originate from mutations in the GlyR gene, leading to a decrease in single channel conductance,
a lower affinity to the neurotransmitter, or a low level of GlyR expression. The function of glycinergic synapses is modulated
during developmental changes and strictly controlled by several feedback mechanisms at pre-and post-synaptic levels. The developmental
modulation consists in switch in the GlyR subunit composition and change in the chloride homeostasis during the synaptic maturation
and formation of inhibitory networks. Retrograde signalling plays an important role in the synaptic function of HMs; it provides
post-synaptic neurons with efficient tools for controlling pre-synaptic afferents. Glycine receptors and glycinergic synapses
are also regulated by intracellular Ca2+. The mechanisms of these modulations are discussed.
Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 338–349, July–October, 2007. 相似文献
14.
Breitinger HG Villmann C Rennert J Ballhausen D Becker CM 《Journal of neurochemistry》2002,83(1):30-36
The human glycine receptor subunit alpha3 exists in two splice variants (alpha3K/L), with alpha3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3-4 loop. Homomeric alpha3K glycine receptors show faster desensitization than alpha3L receptors. Ion channel properties were compared of alpha3L, alpha3K, and of the triple mutant alpha3LDeltaOH = alpha3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site-directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch-clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non-desensitizing current was 68 +/- 13% for alpha3L, 21 +/- 13% for alpha3K, and 48 +/- 16% for alpha3LDeltaOH. Desensitization time constants at saturating glycine concentration were 8.4 +/- 2.8 s, 1.9 +/- 2.3 s, and 2.8 +/- 0.4 s, for alpha3L, alpha3K, and the triple mutant alpha3LDeltaOH, respectively. In contrast, single-channel and whole-cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3-4 loop of the glycine receptor alpha3 subunit are crucial, but not exclusive, determinants of receptor desensitization. 相似文献
15.
We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR. 相似文献
16.
Ion permeation through light-activated channels in rhabdomeric photoreceptors. Role of divalent cations
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《The Journal of general physiology》1996,107(6):715-730
The receptor potential of rhabdomeric photoreceptors is mediated primarily by a Na influx, but other ions must also permeate through light-dependent channels to account for some properties of the photoresponse. We examined ion conduction in macroscopic and single- channel light-induced currents of Lima and Pecten photoreceptors. In the absence of Na, a fivefold change in extracellular K shifted the reversal voltage of the photocurrent (Vrev) by approximately 27 mV. Because the dependency of Vrev on [K]o was sub-Nernstian, and Vrev in each condition was more positive than Ek, some other ion(s) with a positive equilibrium potential must be implicated, in addition to K. We assessed the participation of calcium, an important candidate because of its involvement in light adaptation. Three strategies were adopted to minimize the impairments to cytosolic Ca homeostasis and loss of responsiveness that normally result from the required ionic manipulations: (a) Internal dialysis with Na-free solutions, to prevent reverse operation of the Na/Ca exchanger. (b) Rapid solution changes, temporally limiting exposure to potentially detrimental ionic conditions. (c) Single-channel recording, exposing only the cell- attached patch of membrane to the test solutions. An inward whole-cell photocurrent could be measured with Ca as the only extracellular charge carrier. Decreasing the [Ca]o to 0.5 mM reduced the response by 43% and displaced the reversal potential by -4.3 mV; the shift was larger (delta Vrev = -44 mV) when intracellular permeant cations were also removed. In all cases, however, the current carried by Ca was < 5% of that measured with normal [Na]o. Unitary light-activated currents were reduced in a similar way when the pipette contained only divalent cations, indicating a substantial selectivity for Na over Ca. The fall kinetics of the photoresponse was slower when external Ca was replaced by Ba, or when the membrane was depolarized; however, dialysis with 10 mM BAPTA failed to antagonize this effect, suggesting that mechanisms other than the Ca influx participate in the modulation of the time course of the photocurrent. 相似文献
17.
Ginsenosides, major active ingredients of Panax ginseng, are known to regulate the excitatory ligand-gated ion channel activity. Recent reports showed that ginsenosides attenuate nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides also affect the inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human glycine alpha1 receptor channel activity expressed in Xenopus oocytes using a two-electrode voltage clamp technique. Treatment of ginsenoside Rf enhances glycine-induced inward peak current (IGly) with dose dependent and reversible manner but ginsenoside Rf itself did not elicit membrane currents. The half-stimulatory concentrations (EC50) of ginsenoside Rf was 49.8 +/- 8.9 microM. Glycine receptor antagonist strychnine completely blocked the inward current elicited by glycine plus ginsenoside Rf. Cl- channel blocker 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) also blocked the inward current elicited by glycine plus ginsenoside Rf. We also tested the effect of eight individual ginsenosides (i.e., Rb1, Rb2, Rc, Rd, Re, Rg1, Rg2, and Ro) in addition to ginsenoside Rf. We found that five of them significantly enhanced the inward current induced by glycine with the following order of potency: Rb1 > Rb2 > Rg2 > or = Rc > Rf > Rg1 > Re. These results indicate that ginsenosides might regulate gylcine receptor expressed in Xenopus oocytes and this regulation might be one of the pharmacological actions of Panax ginseng. 相似文献
18.
A study was carried out to electric parameters of single ionic channels initiated at phase transition of bromidmetilate 1,2-distearoyl-rac-glycero-3-(O-beta-dimethylaminoethyl)-methylphosphonate, whose molecules under conditions given below are possibly charged. It has been shown that changes of transmembrane current appear at phase transition temperature. Comparison between ionic selectivity of channels initiated at Tph.t in the membranes of DSL and its phosphate analog suggests that the channel walls initiated at phospholipid phase transitions are covered with polar groups of molecules. 相似文献
19.
J Sadoshima N Akaike H Tomoike H Kanaide M Nakamura 《Comparative biochemistry and physiology. A, Comparative physiology》1989,92(1):61-63
1. Properties of the voltage-dependent anion-selective channel in cultured smooth muscle cells of the rat aorta were studied using the patch-clamp technique. 2. The channel had a single channel conductance of 346 +/- 4 pS (n = 43, mean +/- SEM) with symmetrical 142 mM-Cl- solution in inside-out patch configurations. 3. The channel was activated spontaneously at a potential range -20 approximately +20 mV and inactivated more rapidly with increases to more positive or negative potentials. 4. The channel was selective for anions and the permeability ratio for monovalent anion was Br-:Cl-:HCOO-:CH3COO-:propionate-:aspartate- = 1.1:1:0.7:0.4: less than 0.02: less than 0.02. 5. The openings of the channels were observed more frequently in inside-out membrane patches than in cell-attached ones, and were independent of intracellular free Ca concentrations. 6. The density of this channel was estimated to be 1.3/micron2. 7. Physiological roles of the channel were discussed. 相似文献
20.
Steinacker P Awni LA Becker S Cole T Reymann S Hesse D Kratzin HD Morris-Wortmann C Schwarzer C Thinnes FP Hilschmann N 《The international journal of biochemistry & cell biology》2000,32(2):225-234
Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins. 相似文献