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1.
M. Pusch 《European biophysics journal : EBJ》1990,18(6):317-326
1. Macroscopic and single-channel currents through several types of cloned rat brain Na+ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na+ channels in chromaffin cells, intracellular Mg2+ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na+ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg2+. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg2+ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na]
i
); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg2+ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg]
i
. This indicates that Mg2+ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 108 M–1 s–1 and 3.6 · 105 s–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg2+ strongly depends on [Na]
i
. This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg2+ block at physiological levels of [Mg]
i
.abbreviations [Na]i
intracellular Na+ concentration
- [K]
i
intracellular K+ concentration
- [Mg]
i
intracellular Mg2+ concentration
- HEPES
N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid
- EGTA
ethyleneglycol-bis-[\-amino-ethyl ether] N,N-tetra acetic acid
- TEA
tetraethylammonium 相似文献
2.
Yamaoka K Yuki T Kawase K Munemori M Seyama I 《American journal of physiology. Heart and circulatory physiology》2002,282(3):H1092-H1101
We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes. 相似文献
3.
Viera Kominkova Lubica Malekova Zuzana Tomaskova Peter Slezak Adam Szewczyk Karol Ondrias 《BBA》2010,1797(6-7):1300-1312
We report the effects of ATP and Mg2+ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg2+ decreased the chloride channel current (n = 12/14), whereas Mg2+ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg2+ modulation of these interactions may regulate different physiological and pathological processes. 相似文献
4.
5.
6.
Uwe Ludewig Christoph Lorra Olaf Pongs Stefan H. Heinemann 《European biophysics journal : EBJ》1993,22(4):237-247
The members of the RCK family of cloned voltage-dependent K+ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K+ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg2+ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K+ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg2+ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K+ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K+ and thereby indirectly affects internal Mg2+ block and channel closing.Abbreviations TEA
tetraethylammonium
- EGTA
Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid
- 2S3B model
2-site 3-barrier model
Correspondence to: S. H. Heinemann 相似文献
7.
Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers. 总被引:14,自引:3,他引:11
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We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential. 相似文献
8.
We have investigated the permeability of the Cav3.1 channel for Ca2+ and different monovalent cations and the block of the currents by Mg2+ ions. In the absence of extracellular divalent cations, the Cav3.1 channel was more permeable for Na+ than for Cs+ and impermeable for NMDG+. Monovalent currents were inhibited by Mg2+ of near physiological concentration by three orders of magnitude more effectively than the Ca2+ current. Inhibition of outward, but not inward current by Mg2+ was voltage-dependent. Furthermore, magnesium slowed down channel deactivation presumably by interacting with an open channel state. 相似文献
9.
Regulation of intracellular magnesium by Mg2+ efflux 总被引:1,自引:0,他引:1
T. Günther J. Vormann R. Förster 《Biochemical and biophysical research communications》1984,119(1):124-131
Chicken erythrocytes were loaded with Mg2+ by incubation with the cation ionophore A 23187 in the presence of Mg2+. After removing A 23187 by intensive washing with serum albumin and reincubating the Mg2+-loaded cells, Mg2+ was transported out of the cells until the original Mg2+ content was achieved. The net Mg2+ efflux followed Michaelis-Menten-kinetics and was independent of extracellular and intracellular Ca2+ and calmodulin. The net Mg2+ efflux was not affected by adrenalin, isoproterenol, p-chloromercuribenzenesulfonate, ouabain and tetrodotoxin, but was inhibited by dicyclohexylcarbodiimide, KCN, iodoacetate, high extracellular concentrations of Mg2+, Mn2+ and when extracellular Na+ was substituted by choline or K+. The efflux of 1 Mg2+ was coupled with the uptake of 2 Na+. It is concluded that there exists an additional gating process at the inner cell surface becoming active only at increased concentrations of intracellular free Mg2+ regulating the exit of Mg2+ by the efflux system. 相似文献
10.
N Masumoto K Tasaka J Mizuki A Miyake O Tanizawa 《Biochemical and biophysical research communications》1992,182(2):906-912
Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, 4,4'diisothiocyano-2,2' disulfonic acid stilbene (DIDS). Superoxide dismutase (SOD), injected into cells by cell fusion, also inhibited the change of [Mg2+]i, but catalase did not. Superoxide anion induced prompt increase of intracellular pH (pHi) as well as decrease of [Mg2+]i and subsequently activated the increase of intracellular free Ca2+ ([Ca2+]i) and the release of arachidonate. In contrast to superoxide anion, NH4Cl which induces increase of pHi in amnion cells increased [Mg2+]i. The elevation of basal level of [Mg2+]i by Mg(2+)-ionophore inhibited the change of [Ca2+]i and the release of arachidonate induced by superoxide anion. These results suggest that superoxide anion, transported through anion channels into cells, decreases [Mg2+]i directly, not due to a pH-effect and that the decrease of [Mg2+]i may regulate biological functions of the cells via increase of [Ca2+]i. 相似文献
11.
Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium
The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium. 相似文献
12.
《The Journal of general physiology》1984,84(5):687-704
Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field. 相似文献
13.
Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes 总被引:3,自引:0,他引:3
Droogmans Guy; Prenen Jean; Eggermont Jan; Voets Thomas; Nilius Bernd 《American journal of physiology. Cell physiology》1998,275(3):C646
We have studiedthe effects of calix[4]arenes on the volume-regulated anionchannel (VRAC) currents in cultured calf pulmonary artery endothelialcells. TS- and TS-TM-calix[4]arenes induced a fastinhibition at positive potentials but were ineffective at negativepotentials. Maximal block occurred at potentials between 30 and 50 mV.Lowering extracellular pH enhanced the block and shifted the maximuminhibition to more negative potentials. Current inhibition was alsoaccompanied by an increased current noise. From the analysis of thecalix[4]arene-induced noise, we obtained a single-channelconductance of 9.3 ± 2.1 pS (n = 9) at +30 mV. The voltage- and time-dependent block were describedusing a model in which calix[4]arenes bind to a site at anelectrical distance of 0.25 inside the channel with an affinity of 220 µM at 0 mV. Binding occludes VRAC at moderately positive potentials,but calix[4]arenes permeate the channel at more positivepotentials. In conclusion, our data suggest an open-channel block ofVRAC by calix[4]arenes that also depends on the protonationof the binding site within the pore. 相似文献
14.
Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels 总被引:26,自引:9,他引:17
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The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance. 相似文献
15.
Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS. 相似文献
16.
D Moran 《The American journal of anatomy》1991,192(1):14-22
General models of cell activation implicate Ca2+ conductance as pivotal in conveying transmembrane signals. During embryonic development, both cell migration and differentiation are influenced by changes in Ca2+; and, as a consequence, the modulation of Ca2+ is important in the control of many morphogenetic processes. Because Ca2+ conductance may be regulated at voltage-dependent Ca2+ channels (VD-CCs), we investigated whether neural crest cells develop VDCCs and, if so, whether they function in regulating migration and establishing cytomorphology. Autoradiography indicates that neural crest cells in vitro develop -L-type Ca2+ channels during migration and differentiation. Blockage of these channels by verapamil, both in vivo and in vitro, leads to a dramatic and reversible inhibition of neural crest migration. Alterations are manifest in vitro in cell-to-cell and cell-to-substratum contact and in the organization of the actin cytoskeleton. In whole embryos, verapamil or nifedipine inhibits pigment pattern formation. Moreover, blockage of the -L-type Ca2+ channels in whole embryos or cultures, after cells have already migrated and differentiated, results in a significant change in individual cell shape and in the overall pigment cell pattern, suggesting further that maintenance of the differentiated state also requires regulation at the -L-type Ca2+ channel. Since certain aspects of neural crest adhesion and cytoskeletal function are dependent on Ca2+, it is suggested that interactions that regulate the availability of Ca2+ through the VDCC may provide coordinate control of motile and adhesive interactions at the cell-substratum interface. 相似文献
17.
Tetraethylammonium ion (TEA) and its longer chain derivatives have been used extensively to block currents through K-selective ion channels. Substantial information has been gained about the structure and gating mechanisms of K and other cation channels from the analysis of the blocking interactions of TEA and other quaternary ammonium ions. We now present an analysis of blocking interactions between single Cl-selective ion channels from acutely dissociated rat cortical neurons and externally applied TEA. TEA applied to the extracellular membrane surface (TEAo) blocked Cl channels in a voltage-dependent manner, with hyperpolarizing potentials favoring block. The voltage dependence of block could be adequately fit assuming that TEA enters the channel pore and binds to a site located approximately 28% of the way through the membrane electrical field. The dose-response relationship between fractional current and [TEA]o at a fixed holding potential of -40 mV was well fit to a simple model with two blocking sites with dissociation constants (Kd) of approximately 2 and 70 mM. The dose-response relationship could also be fit by a mechanism where TEA only partially blocks the channels. At the bandwidth used in these experiments (1-2 kHz), both the mean open duration (composed of the open and blocked durations) and burst duration (composed of open, blocked, and short lifetime shut durations) increased with increased [TEA]o. This is expected if TEAo can bind and unbind only when the channel is in the open kinetic state. These results suggest that the structure of the permeability pathway of these anion-selective channels may be very similar to that of other channels that are blocked by TEA. Additionally, these results caution that a blocking effect by TEA cannot, by itself, be used as sufficient evidence for implicating the participation of K channels in a particular process. 相似文献
18.
Isaev D Solt K Gurtovaya O Reeves JP Shirokov R 《The Journal of general physiology》2004,123(5):555-571
Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation. 相似文献
19.
Intracellular blockade by quaternary ammonium (QA) molecules of many potassium channels is state dependent, where the requirement for channel opening is evidenced by a time-dependent component of block in the macroscopic record. Whether this is the case for Ca(2+)- and voltage-activated potassium (BK) channels, however, remains unclear. Previous work (Li, W., and R.W. Aldrich. 2004. J. Gen. Physiol. 124:43-57) tentatively proposed a state-dependent, trapping model, but left open the possibility of state-independent block. Here, we found BK channel blockade by a novel QA derivative, bbTBA, was time dependent, raising the possibility of state-dependent, open channel block. Alternatively, the observed voltage dependence of block could be sufficient to explain time-dependent block. We have used steady-state and kinetic measurements of bbTBA blockade in order to discriminate between these two possibilities. bbTBA did not significantly slow deactivation kinetics at potentials between -200 and -100 mV, suggesting that channels can close unhindered by bound bbTBA. We further find no evidence that bbTBA is trapped inside BK channels after closing. Measurements of steady state fractional block at +40 mV revealed a 1.3-fold change in apparent affinity for a 33-fold change in P(o), in striking contrast to the 31-fold change predicted by state-dependent block. Finally, the appearance of a third kinetic component of bbTBA blockade at high concentrations is incompatible with state-dependent block. Our results suggest that access of intracellular bbTBA to the BK channel cavity is not strictly gated by channel opening and closing, and imply that the permeation gate for BK channels may not be intracellular. 相似文献
20.
Functional assays of intracellular Ca2+ channels, such as the inositol 1,4,5-trisphosphate receptor (IP3R), have generally used 45Ca2+-flux assays, fluorescent indicators loaded within either the cytosol or the endoplasmic reticulum (ER) of single cells, or electrophysiological analyses. None of these methods is readily applicable to rapid, high-throughput quantitative analyses. Here we provide a detailed protocol for high-throughput functional analysis of native and recombinant IP3Rs. A low-affinity Ca2+ indicator (mag-fluo-4) trapped within the ER of permeabilized cells is shown to report changes in luminal free Ca2+ concentration reliably. An automated fluorescence plate reader allows rapid measurement of Ca2+ release from intracellular stores mediated by IP3R. The method can be readily adapted to other cell types or to the analysis of other intracellular Ca2+ channels. This protocol can be completed in 2-3 h. 相似文献