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1.
The temperature-dependent ion conductance of OmpC, a major outer membrane channel of Escherichia coli, is predicted using all-atom molecular dynamics simulations and experimentally verified. To generalize previous results, OmpC is compared to its structural homolog OmpF at different KCl concentrations, pH values, and a broad temperature range. At low salt concentrations and up to room temperature, the molecular modeling predicts the experimental conductance accurately. At high salt concentrations above 1 M KCl and above room temperature, the simulations underestimate the conductance. Moreover, the temperature dependence of the channel conductance is different from that of the bulk, both in experiment and simulation, indicating a strong contribution of surface effects to the ion conductance. With respect to OmpC, subconductance levels can be observed in experiments only. Subconductance and gating levels can be clearly distinguished by their differences in conductance values and temperature-dependent behavior. With increasing temperature, the probability of a subconductance state to occur, increases, while the dwell time is decreased. The open probability, frequency, and dwell time of such states is largely pH- and KCl concentration-independent, while their amplitudes show a lower increase with increasing salt concentration than gating amplitudes. Voltage dependence of subconductance has been found to be negligible within the uncertainty of the measurements.  相似文献   

2.
The temperature dependence of the properties of unitary currents in cultured rat ventricular myocytes has been studied. Currents flowing through an ATP-dependent K+ channel were recorded from inside-out patches with the bath temperature varied from 10 degrees to 30 degrees C. The channel conductance was 56 pS at room temperature (22 degrees C), and the amplitudes of unitary currents and the channel conductance exhibited a relatively weak (Q10 from 1.4 to 1.6) dependence on temperature. The temperature dependence of channel mean open times was biphasic with the low temperature (10-20 degrees C) range showing a relatively stronger temperature dependence (Q10 of 2.3) than the high temperature (20-30 degrees C) range (Q10 of 1.6). The activation energies for the two regions were determined from an Arrhenius plot with the activation energy, corresponding to the lower temperature range, near 16 kcal/mol. Thermodynamic analysis, using transition rate theory, indicated that the formation of a transition state prior to channel closure to be associated with a positive entropy component for the high Q10 region.  相似文献   

3.
The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed.  相似文献   

4.
The temperature and voltage dependence of gating and conductance of sarcoplasmic reticulum K+ channels (S-R K+) isolated from adult canine hearts were studied using the reconstituted bilayer technique. Fusion of vesicles from this preparation frequently resulted in the incorporation of a single channel. Only bilayers into which a single S-R K+ channel had fused were studied. The three conductance states of the channel, fully open (O2), substate conductance (O1), and closed (C) were studied as a function of voltage (-50 to +50 mV) and temperature (16 to 37 degrees C). Permeation through the O1 state showed the same temperature dependence as the O2 state corresponding to an enthalpy of permeation of 4.1-4.2 kcal/mol, which is similar to that for K+ diffusion through water. As expected, increased temperature increased the frequency of gating transitions and shortened the average dwell time spent in any conductance state. Over the range of 25 to 37 degrees C, the average dwell time spent in the O1, O2, and C states decreased by 44 +/- 11, 36 +/- 13, and 78 +/- 7% (n = 3 to 4 channels), respectively. The ratio of probabilities between the various conductance states was not strongly temperature sensitive. Analysis of the voltage dependence of this channel was carried out at 37 degrees C and revealed that the dwell times of the O1 and O2 states were voltage insensitive and the probability ratio (PO2:PO1) was approximately 7 and was voltage insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
Transport at the nanoscale: temperature dependence of ion conductance   总被引:1,自引:0,他引:1  
Temperature dependent ion conductance in nanopores is measured in a wide range of electrolyte concentrations and compared with molecular modeling. Single outer membrane protein F (OmpF) channels from E. coli are reconstituted into planar lipid bilayers. In qualitative agreement with the experimental data, applied-field molecular dynamics unraveled atomistic details of the ion transport. Comparing the temperature dependence of the channel conductance with that of the bulk conductivity in the range from 0 to 90°C revealed that at low salt concentrations the transport is mainly driven along the pore surface. Increasing the salt concentration saturates the surface charge transport and induces ion transport in the center of the nanopore. The confinement of the nanopore then favors the formation of ion pairs. Stepping up the temperature reduces the life time of the ion pairs and increases the channel conductance more than expected from the bulk behavior.  相似文献   

6.
Summary A study of the temperature dependence of gramicidin A conductance of K+ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance, states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation,vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.  相似文献   

7.
The effects of microwaves on the single-channel kinetics of gramicidin-A channels in lipid bilayer membranes were examined. Attempts were made to separate thermal and athermal effects by accurate measurements of temperature at the site of the membrane and by relating the measured parameters to their previously characterized temperature dependence. It was found that microwave radiation does not affect single-channel conductance or channel life time to a degree that is significantly different from that expected of a purely thermal effect. On the other hand, the rate of channel formation is decreased during exposure, which is opposite to that expected of a purely thermal effect. The mechanism of this effect is discussed in terms of the dimerization process of channel formation.  相似文献   

8.
We have reported that the large impermeant organic cations tetrabutyl ammonium (TBA+), tetrapentyl ammonium, and the charged local anesthetic QX314 produce unique reduced conductance states in the purified sheep cardiac sarcoplasmic reticulum Ca2+ release channel when present at the cytoplasmic face of the channel. We have interpreted this as a form of partial occlusion by the blocking cation in wide vestibules of the conduction pathway. Following modification with ryanodine, which causes the channel to enter a reduced conductance state with long open dwell time, these cations block the receptor channel to a level that is indistinguishable from the closed state. The voltage dependence of TBA+'s interaction with the Ca2+ release channel is the same before and after ryanodine modification. The concentration dependence is different, in that the ryanodine-modified channel has one-third the affinity for TBA+, which is accounted for predominantly by changes in the TBA+ on rate. The data are compatible with a structural change in the vestibule of the conduction pathway consequent upon ryanodine binding that reduces the capture radius for blocking ion entry.  相似文献   

9.
Channel access resistance has been measured to estimate the characteristic size of a single ion channel. We compare channel conductance in the presence of nonpenetrating water-soluble polymers with that obtained for polymer-free electrolyte solution. The contribution of the access resistance to the total alamethicin channel resistance is approximately 10% for first three open channel levels. The open alamethicin channel radii inferred for these first three levels from the access resistance are 6.3, 10.3, and 11.4 A. The dependence of channel conductance on polymer molecular weight also allows evaluation of the channel dimensions from polymer exclusion. Despite varying conductance, it was shown that steric radii of the alamethicin channel at different conductance levels remain approximately unchanged. These results support a model of the alamethicin channel as an array of closely packed parallel pores of nearly uniform diameter.  相似文献   

10.
N-methyl-D-aspartate (NMDA) receptor channels in cultured CA1 hippocampal neurons were studied using patch-clamp techniques. The purpose of the research was to determine the occupancy of the channel by permeant cations and to determine the influence of charged residues in or near the pore. The concentration dependence of permeability ratios, the mole-fraction dependence of permeability ratios, the concentration dependence of the single-channel conductance, and a single-channel analysis of Mg2+ block all independently indicated that the NMDA receptor behaves as a singly-occupied channel. More precisely, there is one permeant cation at a time occupying the site or sites that are in the narrow region of the pore directly in the permeation pathway. Permeability-ratio measurements in mixtures of monovalent and divalent cations indicated that local charges in or near the pore do not produce a large local surface potential in physiologic solutions. In low ionic strength solutions, a local negative surface potential does influence the ionic environment near the pore, but in normal physiologic solutions the surface potential appears too small to significantly influence ion permeation. The results indicate that the mechanism for the high Ca2+ conductance of the NMDA receptor channel is not the same as for the voltage-dependent Ca2+ channel (VDCC). The VDCC has two high affinity, interacting binding sites that provide high Ca2+ selectivity and conductance. The binding site of the NMDA receptor is of lower affinity. Therefore, the selectivity for Ca2+ is not as high, but the lower affinity of binding provides a faster off rate so that interacting sites are not required for high conductance.  相似文献   

11.
By using single-channel recording techniques, we measured the conductance (gK) of the Ca(2+)-activated Maxi-K+ channel from the embryonic rat brain, and examined its dependence on K+ ions present in equimolar concentrations on both sides of the membrane patch. With ionic strength maintained constant by substitution of N-methyl-D-glucamine for K+, gK has a sigmoidal dependence upon [K+]. This result has been obscured in previous work by variations in ionic strength, which has a marked effect on single-channel conductance, especially in the limit for which this variable approaches zero. The gK versus [K+] relationship is described, theoretically, by a three-barrier, two-binding-site model in which the barrier that an ion must cross to leave the channel is decreased as [K+] is increased.  相似文献   

12.
Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.  相似文献   

13.
The binding component (Vip1Ac) of the ADP-ribosylating vegetative insecticidal protein (Vip) of Bacillus thuringiensis HD201 was isolated from the supernatant of cell cultures. Vip1Ac protein solubilized at room temperature ran as oligomers on SDS-PAGE. These oligomers were not resistant to heating. Mass spectroscopic analysis of this high molecular mass band identified it as Vip1Ac. The protein formed in artificial lipid bilayer membranes channels with two conductance states of about 350 and 700 pS in 1 M KCl. The channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero-current membrane potential measurements showed that the Vip1Ac channel has a slightly higher permeability for chloride than for potassium ions. Asymmetric addition of Vip1Ac to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. The functional role of Vip1Ac and its relationship to other ADP-ribosylating toxins are discussed.  相似文献   

14.
Ilan N  Moran N  Schwartz A 《Plant physiology》1995,108(3):1161-1170
We used the patch-clamp technique to examine the effect of temperature (13-36[deg]C) on the depolarization-activated K channels (KD channels) and on the hyperpolarization-activated channels (KH channels) in the plasma membrane of Vicia faba guard-cell protoplasts. The steady-state whole-cell conductance of both K channel types increased with temperature up to 20[deg]C. However, whereas the whole-cell conductance of the KH channels increased further and saturated at 28[deg]C, that of KD channels decreased at higher temperatures. The unitary conductance of both channel types increased with temperature like the rate of diffusion in water (temperature quotient of approximately 1.5), constituting the major contribution to the conductance increase in the whole cells. The mean number of available KH channels was not affected significantly by temperature, but the mean number of available KD channels increased significantly between 13 and 20[deg]C and declined drastically above 20[deg]C. This decrease and the reduced steady-state voltage-dependent probability of opening of the KD channels above 28[deg]C (because of a shift of voltage dependence by +21 mV) account for the depression of the whole-cell KD conductance at the higher temperatures. This may be a basic mechanism by which leaves of well-watered plants keep their stomata open during heat stress to promote cooling by transpiration.  相似文献   

15.
The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.  相似文献   

16.
The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca(2+) blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca(2+) block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca(2+) that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca(2+) affinity are active.  相似文献   

17.
The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membranes depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).  相似文献   

18.
Temperature dependence of ion permeation at the endplate channel   总被引:4,自引:1,他引:3       下载免费PDF全文
The dependence of acetylcholine receptor mean single-channel conductance on temperature was studied at garter snake twitch-muscle endplates using fluctuation analysis. In normal saline under conditions where most of the endplate current was carried by Na+, the channel conductance increased continuously from near 0 degrees C to approximately 23 degrees C with a Q10 of 1.97 +/- 0.14 (mean +/- SD). When 50% of the bath Na+ was replaced by either Li+, Rb+, or Cs+, the Q10 did not change significantly; however, at any temperature the channel conductance was greatest in Cs-saline and decreased with the ion sequence Cs greater than Rb greater than Na greater than Li. The results were fit by an Eyring-type model consisting of one free-energy well on the extracellular side of a single energy barrier. Ion selectivity appeared to result from ion-specific differences in the well and not in the barrier of this model. With a constant barrier enthalpy for different ions, well free-energy depth was greatest for Cs+ and graded identical to the permeability sequence. The correlation between increased well depth (i.e., ion binding) and increased channel conductance can be accounted for by the Boltzmann distribution of thermal energy.  相似文献   

19.
Ion channels activated by light in Limulus ventral photoreceptors   总被引:6,自引:5,他引:1  
The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors.  相似文献   

20.
We have identified in organic solvent extracts of whole cells of the gram-positive pathogen Rhodococcus equi two channel-forming proteins with different and complementary properties. The isolated proteins were able to increase the specific conductance of artificial lipid bilayer membranes made from phosphatidylcholine-phosphatidylserine mixtures by the formation of channels able to be permeated by ions. The channel-forming protein PorA(Req) (R. equi pore A) is characterized by the formation of cation-selective channels, which are voltage gated. PorA(Req) has a single-channel conductance of 4 nS in 1 M KCl and shows high permeability for positively charged solutes because of the presence of negative point charges. According to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein has an apparent molecular mass of about 67 kDa. The analysis (using the effect of negative charges on channel conductance) of the concentration dependence of the single-channel conductance suggested that the diameter of the cell wall channel is about 2.0 nm. The second channel (formed by PorB(Req) [R. equi pore B]) shows a preferred movement of anions through the channel and is not voltage gated. This channel shows a single-channel conductance of 300 pS in 1 M KCl and is characterized by the presence of positive point charges in or near the channel mouth. Based on SDS-PAGE, the apparent molecular mass of the channel-forming protein is about 11 kDa. Channel-forming properties of the investigated cell wall porins were compared with those of others isolated from mycolic acid-containing actinomycetes. We present here the first report of a fully characterized anion-selective cell wall channel from a member of the order Actinomycetales.  相似文献   

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