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1.
Inhibition of Vacuolar Ion Channels by Polyamines 总被引:1,自引:0,他引:1
In this work, direct effects of cytosolic polyamines on the two principle vacuolar ion channels were studied by means of
patch-clamp technique. Fast and slow activating vacuolar channels were analyzed on membrane patches isolated from vacuoles
of the red beet taproot. The potency of the fast and of the slow vacuolar channel blockage by polyamines decreased with a
decrease of the polycation charge, spermine4+ > spermidine3+ > putrescine2+. In contrast to the inhibition of the fast vacuolar channel, the blockage of the slow vacuolar channel by polyamines displayed
a pronounced voltage-dependence. Hence, in the presence of high concentration of polyamines the slow vacuolar channel was
converted into a strong inward rectifier as evidenced by its unitary current-voltage characteristic. The blockage of the slow
vacuolar channel by polyamines was relieved at a large depolarization, in line with the permeation of polyamines through this
channel. The voltage-dependence of blockage was analyzed in terms of the conventional model, assuming a single binding site
for polyamines within the channel pore. Taking advantage of a simple linear structure of naturally occurring polyamines, conclusions
on a possible architecture of the slow vacuolar channel pore were drawn. The role of common polyamines in regulation of vacuolar
ion transport was discussed.
Received: 1 May 1998/Revised: 25 September 1998 相似文献
2.
Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked
glycosylation of voltage-gated K+ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single
consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide
chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern
of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating
oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains
on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels,
reflecting the different efficiencies of transport of K+ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich
N-linked oligosaccharide chains differs among highly related K+ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential
glycosylation contributing to diversity of K+ channel function in mammalian brain.
Received: 17 December 1998/Accepted: 20 January 1999 相似文献
3.
Ion channel activity in cell-attached patch recordings shows channel behavior under more physiological conditions than whole-cell
and excised patch measurements. Yet the analysis of cell-attached patch measurements is complicated by the fact that the system
is ill defined with respect to the intracellular ion activities and the electrical potential actually experienced by the membrane
patch. Therefore, of the several patch-clamp configurations, the information that is obtained from cell-attached patch measurements
is the most ambiguous. The present study aims to achieve a better understanding of cell-attached patch measurements. Here
we describe a method to calculate the intracellular ion concentration and membrane potential prevailing during cell-attached
patch recording. The first step is an analysis of the importance of the input resistance of the intact cell on the cell-attached
patch measurement. The second step, and actual calculation, is based on comparison of the single channel conductance and reversal
potential in the cell-attached patch and excised patch configurations. The method is demonstrated with measurements of membrane
potential and cytosolic K+ concentrations in Vicia faba guard cells. The approach described here provides an attractive alternative to the measurement of cytosolic ion concentrations
with fluorescent probes or microelectrodes.
Received: 3 April 1998/Revised: 6 August 1998 相似文献
4.
The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K+, Na+, Ca2+ and Ba2+ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model),
which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy
profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles
in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak
differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred
within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current
vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary
conductance vs. activity relationships for K+, Na+ and Ca2+, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative
cation permeabilities with solution composition, and the complex effects of Ba2+ and Ca2+ on K+ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation
channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input
to electrocoupling models allowing the relationships between membrane voltage, Ca2+ influx and Ca2+ signaling to be studied theoretically.
Received: 29 April 1998/Revised: 20 November 1998 相似文献
5.
Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to
patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses,
which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols
revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude,
providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml
pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation.
Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation
consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these
data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated
protein.
Received: 29 January 1998/Revised: 16 April 1998 相似文献
6.
Theoretical evaluation of cell membrane ion channel activation by applied magnetic fields 总被引:5,自引:0,他引:5
This letter re-examines a recently published calculation of the forces exerted on a membrane ion channel by a cation passing
through in the presence of an externally applied magnetic field. We show here, in contradiction to the originally published
calculation, that the forces generated due to the Lorentz force of the magnetic field on the cation are negligible compared
with the forces required to activate an ion channel protein conformation change associated with the gating of the channel.
Received: 11 August 1998 / Revised version: 25 October 1998 / Accepted: 11 November 1998 相似文献
7.
Temperature was used as a biophysical tool to investigate the energy changes associated with conformational change during
the gating of a non-inactivating voltage-gated K+ channel present in the membrane of αT3-1 cells, a gonadotroph cell line. The time course of the current activation was described by a single exponential function
at three temperatures: 15, 25 and 35 °C. The Q
10 values were between 1.5 to 1.9 and in agreement with the activation energy determined from Arrhenius plots of the forward
and backward rate constants associated with channel opening. The Gibb's free energy change associated with channel opening
and closing at various membrane potentials estimated by two approaches yield similar values. The changes in Gibb's free energy
(ΔG°) with depolarization potential is a quadratic and more prominent at 15 than at 25 or 35 °C. The results suggest that increase
in temperature favours movement of voltage sensing segments, and reduces the restraint on them brought about by other parts
of the channel molecule.
Received: 2 September 1998 / Revised version: 27 October 1998 / Accepted: 21 January 1999 相似文献
8.
The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different
ions in the external medium. With either K+, NH+
4 or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K+ and, to a lesser extent, to NH+
4 but not to the slightly bigger, methylated analogue of NH+
4, MA. Substituting NH+
4 for K+ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that
channel operation depends on the transported substrate. In mixed solution (50 mm K+, 50 mm MA) MA inhibited K+ current in a voltage-independent manner. The maximum block did not exceed 50% of the K+ current. In contrast, when NH+
4 was the permeant ion (50 mm NH+
4, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages.
The latter block could be partially overcome by the addition of K+ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different
affinities for binding sites on their permeation pathway.
Received: 6 October 1997/Revised: 28 January 1998 相似文献
9.
In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level
of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied
to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the
electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds
for all three types of ion channels investigated: the large-conductance Ca2+-dependent K+ channel (maxi-KCa), a K+ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments
show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before
the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous
reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically
induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter
process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance
also allow permeation of Ca2+ ions. The resulting Ca2+-flux may contribute to the photodynamically induced increase of the intracellular Ca2+ concentration observed in various cell lines.
Received: 26 May 1998/Revised: 8 September 1998 相似文献
10.
11.
Two channels, distinguished by using single-channel patch-clamp, carry out potassium transport across the red cell membrane
of lamprey erythrocytes. A small-conductance, inwardly rectifying K+-selective channel was observed in both isotonic and hypotonic solutions (osmolarity decreased by 50%). The single-channel
conductance was 26 ± 3 pS in isotonic (132 mm K+) solutions and 24 ± 2 pS in hypotonic (63 mm K+) solutions. No outward conductance was found for this channel, and the channel activity was completely inhibited by barium.
Cell swelling activated another inwardly rectifying K+ channel with a larger inward conductance of 65 pS and outward conductance of 15 pS in the on-cell configuration. In this
channel, rectification was due to the block of outward currents by Mg2+ and Ca2+ ions, since when both ions were removed from the cytosolic side in inside-out patches the conductance of the channel was
nearly ohmic. In contrast to the small-conductance channel, the swelling-activated channel was observed also in the presence
of barium in the pipette. Neither type of channel was dependent on the presence of Ca2+ ions on the cytosolic side for activity.
Received: 18 July 1997/Revised: 30 January 1998 相似文献
12.
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. 相似文献
13.
The effects of aldosterone and vasopressin on Cl− transport were investigated in a mouse cortical collecting duct (mpkCCD) cell line derived from a transgenic mouse carrying
the SV40 large T antigen driven by the proximal regulatory sequences of the L-pyruvate kinase gene. The cells had features
of a tight epithelium and expressed the amiloride-sensitive sodium channel and the cystic fibrosis transmembrane conductance
regulator (CFTR) genes. dD-arginine vasopressin (dDAVP) caused a rapid, dose-dependent, increase in short-circuit current
(I
sc
). Experiments with ion channel blockers and apical ion substitution showed that the current represented amiloride-sensitive
Na+ and 5-nitro-2-(3-phenylpropylamino)benzoate-sensitive and glibenclamide-sensitive Cl− fluxes. Aldosterone (5 × 10−7
m for 3 or 24 hr) stimulated I
sc
and apical-to-basal 22Na+ flux by 3-fold. 36Cl− flux studies showed that dDAVP and aldosterone stimulated net Cl− reabsorption and that dDAVP potentiated the action of aldosterone on Cl− transport. Whereas aldosterone affected only the apical-to-basal 36Cl− flux, dDAVP mainly increased the apical-to-basal Cl− flux and the basal-to-apical flux of Cl− to a lesser extent. These results suggest that the discrete dDAVP-elicited Cl− secretion involves the CFTR and that dDAVP and aldosterone may affect in different ways the observed increased Cl− reabsorption in this model of mouse cultured cortical collecting duct cells.
Received: 8 January 1998/Revised: 25 March 1998 相似文献
14.
Formation of plasmid DNA strand breaks induced by low-energy ion beam: indication of nuclear stopping effects 总被引:3,自引:0,他引:3
Y. Chen Bingyao Jiang Youshan Chen Xingzhao Ding Xianghuai Liu Ceshi Chen Xinyou Guo Guanglin Yin 《Radiation and environmental biophysics》1998,37(2):101-106
Plasmid pGEM 3zf(+) was irradiated by nitrogen ion beam with energies between 20 and 100 keV and the fluence kept as 1×1012 ions/cm2. The irradiated plasmid was assayed by neutral electrophoresis and quantified by densitometry. The yields of DNA with single-strand
and double-strand breaks first increased then decreased with increasing ion energy. There was a maximal yield value in the
range of 20–100 keV. The relationship between DNA double-strand breaks (DSB) cross-section and linear energy transfer (LET)
also showed a peak-shaped distribution. To understand the physical process during DNA strand breaks, a Monte Carlo calculation
code known as TRIM (Transport of Ions in Matter) was used to simulate energy losses due to nuclear stopping and to electronic
stopping. It can be assumed that nuclear stopping plays a more important role in DNA strand breaks than electronic stopping
in this energy range. The physical mechanisms of DNA strand breaks induced by a low-energy ion beam are also discussed.
Received: 30 July 1997 / Accepted in revised form: 18 January 1998 相似文献
15.
V.M. Baizabal-Aguirre S. Clemens N. Uozumi J.I. Schroeder 《The Journal of membrane biology》1999,167(2):119-125
The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K+ (K+
in
) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K+
in
channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were
studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K+ currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for
wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not
greatly affect the ion selectivity of residual currents for Rb+, Na+, Li+, or Cs+. When T256R or G262K were co-expressed with a different K+ channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs+ block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers.
These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued
in vivo by the expression of the dominant negative K
+
in
channel mutants described here.
Received: 2 July 1998/Revised: 23 October 1998 相似文献
16.
A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior.
The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although
variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained
depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However,
these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or
became small in Ca2+-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward
current under conditions where K+ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Sr2+, Mg2+, or Mn2+, and was lost upon replacing with equimolar concentration of Ni2+. On the other hand, the cold-induced inward current was not affected significantly by Ba2+, or Sr2+, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations
of Mg2+, Mn2+, or Ni2+. These results indicate that Paramecium cells have heat-activated Ca2+ channels and cold-activated Ca2+ channels and that the cold-activated Ca2+ channel is different from the heat-activated Ca2+ channel in the ion selectivity and the calcium-dependent inactivation.
Received: 9 September 1998/Revised: 22 January 1999 相似文献
17.
Summary The Ca-inward current ofParamecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba2+ increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec–1, or about 1.2 ·108 molecules/cell·sec–1. Because no other factors than the Ca-inward current were found to affect cGMP formation inParamecium, we used it as a quantitative measure of Ca2+ channel activity. After a transient stimulation of cGMP formation by 1mm Ba2+, an additional increase of Ba2+ to 5mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1mm Ba2+ revealed a time-dependent inactivation of the Ca2+ channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization ofParamecium reduced the maximal response, but did not shift the dose-response curve for Ba2+. Veratridine, which activates the Ca2+ channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response ofParamecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol.The extremely high rate of cGMP turnover inParamecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel ofParamecium is proposed. 相似文献
18.
J.I. Kourie 《The Journal of membrane biology》1999,167(1):73-83
The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca2+ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of
the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH
cis
) and luminal pH (pH
trans
) was investigated using the lipid bilayer-vesicle fusion technique. Low pH
cis
6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH
cis
7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas
at low pH
cis
6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low
pH
trans
4.07, but not pH
trans
6.28, modified the activity of SCl channels. The effects of low pH
cis
are pronounced at 10−3 and 10−4
m [Ca2+]
cis
but are not apparent at 10−5
m [Ca2+]
cis
, where the subconductances of the channel are already prominent. Low pH
cis
-induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of
the subconductance states. The results in this study suggest that low pH
cis
can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction
in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels.
Received: 20 May 1998/Revised: 24 September 1998 相似文献
19.
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
20.
Fatty acids play an important role in a variety of physiological processes including ion channel modulation and catecholamine
release. Using patch-clamp techniques we show that arachidonic acid (AA) is converted to lipoxygenase metabolites (LOMs) to
potentiate activity of the Ca2+ and voltage-dependent, large-conductance K+ channel (BK) in bovine adrenal medullary chromaffin cells (BAMCCs). AA and LOM potentiation of BK current and recovery from
potentiation were unaffected by the nonhydrolyzable ATP analogue AMP-PNP, or by exclusion of nucleotides in excised patch
recordings. Also, AA and LOM potentiation of BK channel activity in outside-out patches exposed to strong Ca2+ buffering ruled out cytoplasmic messengers or changes in intracellular Ca2+ levels as causative factors. Lipoxygenase inhibitor attenuated AA, but not LOM potentiation of BK activity in outside-out
patches, indicating that lipoxygenase processing of AA is possible in excised membrane patches, possibly via a membrane associated
lipoxygenase. AA and LOM release have been implicated in the mechanics of catecholamine secretion from BAMCCs. By limiting
action potential duration and thus voltage-gated Ca2+ influx, fatty acid potentiation of BK current may serve an inhibitory feedback function in regulating secretion from BAMCCs.
Received: 15 July 1997/Revised: 27 January 1997 相似文献