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1.
Ionic currents through sodium channels in nodal membranes were measured under voltage clamp conditions both at normal and at low (4.8-4.9) external solution pH. The measurements of so-called 'instantaneous' currents were used to distinguish between the proton blockage in open channels and the influence of low pH on channel gating processes. It is shown that the amount of the proton blockage in open channels decreases as membrane potential becomes more positive. This result suggests that at least one of the acid groups accessible from the outside is located within the conducting pore. The influence of the other group(s) on the degree of potential-dependence of proton blockage is discussed. 相似文献
2.
P Gavazzo C Picco A Menini 《Proceedings. Biological sciences / The Royal Society》1997,264(1385):1157
We have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK1 of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel. 相似文献
3.
Extracellular proton-modulated pore-blocking effect of the anticonvulsant felbamate on NMDA channels
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Felbamate (FBM) is a potent nonsedative anticonvulsant whose clinical effect is chiefly related to gating modification (and thus use-dependent inhibition) rather than pore block of N-methyl-D-aspartate (NMDA) channels at pH 7.4. Using whole-cell recording in rat hippocampal neurons, we examined the effect of extracellular pH on FBM action. In sharp contrast to the findings at pH 7.4, the inhibitory effect of FBM on NMDA currents shows much weakened use-dependence at pH 8.4. Moreover, FBM neither accelerates the activation kinetics of the NMDA channel, nor enhances the currents elicited by very low concentrations of NMDA at pH 8.4. These differential effects of FBM between pH 7.4 and 8.4 are abolished in the mutant NMDA channels which lack proton sensitivity. Most interestingly, the inhibitory effect of FBM becomes flow-dependent and is evidently stronger in inward than in outward NMDA currents at pH 8.4. These findings indicate that FBM has a significantly more manifest pore-blocking effect on the NMDA channel at pH 8.4 than at pH 7.4. FBM therefore acts as an opportunistic pore blocker modulated by extracellular proton, suggesting that the FBM binding site is located at the junction of a widened and a narrow part of the ion conduction pathway. Also, we find that the inhibitory effect of FBM on NMDA currents is antagonized by external but not internal Na+, and that increase of external Na+ decreases the binding rate without altering the unbinding rate of FBM. These findings indicate that the FBM binding site faces the extracellular rather than the intracellular solution, and coincides with the outmost ionic (e.g., Na+) site in the NMDA channel pore. We conclude that the FBM binding site very likely is located in the external pore mouth, where extracellular proton, Na+, FBM, and NMDA channel gating have an orchestrating effect. 相似文献
4.
Yang Z Xu H Cui N Qu Z Chanchevalap S Shen W Jiang C 《The Journal of general physiology》2000,116(1):33-45
CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons. 相似文献
5.
Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H(+)(o)-induced current inhibition by taking advantage of Na(+) permeation through inactivated channels. In hKv1.5, H(+)(o) inhibited open-state Na(+) current with a similar potency to K(+) current, but had little effect on the amplitude of inactivated-state Na(+) current. In support of inactivation as the mechanism for the current reduction, Na(+) current through noninactivating hKv1.5-R487V channels was not affected by [H(+)(o)]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H(+)(o). These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na(+) currents but the maintained presence of slow Na(+) tail currents, combined with changes in the Na(+) tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na(+) current at low pH. 相似文献
6.
Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers. 总被引:1,自引:1,他引:0
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Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers. 相似文献
7.
Modulation by internal protons of native cyclic nucleotide-gated channels from retinal rods 总被引:1,自引:0,他引:1
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Ion channels directly activated by cyclic nucleotides are present in the plasma membrane of retinal rod outer segments. These channels can be modulated by several factors including internal pH (pH(i)). Native cyclic nucleotide-gated channels were studied in excised membrane patches from the outer segment of retinal rods of the salamander. Channels were activated by cGMP or cAMP and currents as a function of voltage and cyclic nucleotide concentrations were measured as pH(i) was varied between 7.6 and 5.0. Increasing internal proton concentrations reduced the current activated by cGMP without modifying the concentration (K(1/2)) of cGMP necessary for half-activation of the maximal current. This effect could be well described as a reduction of single-channel current by protonation of a single acidic residue with a pK(1) of 5.1. When channels were activated by cAMP a more complex phenomenon was observed. K(1/2) for cAMP decreased by increasing internal proton concentration whereas maximal currents activated by cAMP increased by lowering pH(i) from 7.6 to 5.7-5.5 and then decreased from pH(i) 5.5 to 5.0. This behavior was attributed both to a reduction in single-channel current as measured with cGMP and to an increase in channel open probability induced by the binding of three protons to sites with a pK(2) of 6. 相似文献
8.
Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH
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We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels. 相似文献
9.
E A Negulyaev YuAVedernikova 《General physiology and biophysics》1985,4(4):359-365
Ionic currents through fast sodium channels in the neuronal somatic membrane were measured under voltage clamp conditions using external solutions of normal and low pH. Voltage-dependent inhibition of ionic currents through open channels was observed in acidic solutions. The voltage-dependent block of sodium channels may be explained by the presence of two acid groups at the channel. The parameters of the inner and outer acid groups calculated according to this model are similar to those reported for the nodal membrane. 相似文献
10.
Murphy R Cherny VV Morgan D DeCoursey TE 《American journal of physiology. Lung cellular and molecular physiology》2005,288(2):L398-L408
Voltage-gated proton channels are expressed highly in rat alveolar epithelial cells. Here we investigated whether these channels contribute to pH regulation. The intracellular pH (pH(i)) was monitored using BCECF in cultured alveolar epithelial cell monolayers and found to be 7.13 in nominally HCO(3)(-)-free solutions [at external pH (pH(o)) 7.4]. Cells were acid-loaded by the NH(4)(+) prepulse technique, and the recovery was observed. Under conditions designed to eliminate the contribution of other transporters that alter pH, addition of 10 microM ZnCl(2), a proton channel inhibitor, slowed recovery about twofold. In addition, the pH(i) minimum was lower, and the time to nadir was increased. Slowing of recovery by ZnCl(2) was observed at pH(o) 7.4 and pH(o) 8.0 and in normal and high-K(+) Ringer solutions. The observed rate of Zn(2+)-sensitive pH(i) recovery required activation of a small fraction of the available proton conductance. We conclude that proton channels contribute to pH(i) recovery after an acid load in rat alveolar epithelial cells. Addition of ZnCl(2) had no effect on pH(i) in unchallenged cells, consistent with the expectation that proton channels are not open in resting cells. After inhibition of all known pH regulators, slow pH(i) recovery persisted, suggesting the existence of a yet-undefined acid extrusion mechanism in these cells. 相似文献
11.
Víctor De-la-Rosa Esteban Suárez-Delgado Gisela E. Rangel-Yescas León D. Islas 《The Journal of general physiology》2016,147(2):127-136
Proton channels have evolved to provide a pH regulatory mechanism, affording the extrusion of protons from the cytoplasm at all membrane potentials. Previous evidence has suggested that channel-mediated acid extrusion could significantly change the local concentration of protons in the vicinity of the channel. In this work, we directly measure the proton depletion caused by activation of Hv1 proton channels using patch-clamp fluorometry recordings from channels labeled with the Venus fluorescent protein at intracellular domains. The fluorescence of the Venus protein is very sensitive to pH, thus behaving as a genetically encoded sensor of local pH. Eliciting outward proton currents increases the fluorescence intensity of Venus. This dequenching is related to the magnitude of the current and not to channel gating and is dependent on the pH gradient. Our results provide direct evidence of local proton depletion caused by flux through the proton-selective channel. 相似文献
12.
Proton block of unitary currents through BK channels was investigated with single-channel recording. Increasing intracellular proton concentration decreased unitary current amplitudes with an apparent pKa of 5.1 without discrete blocking events, indicating fast proton block. Unitary currents recorded at pH(i) 8.0 and 9.0 had the same amplitudes, indicating that 10(-8) M H(+) had little blocking effect. Increasing H(+) by recording at pH(i) 7.0, 6.0, and 5.0 then reduced the unitary currents by 13%, 25%, and 53%, respectively, at +200 mV. Increasing K(+)(i) relieved the proton block in a manner consistent with competitive inhibition of K(+)(i) action by H(+)(i). Proton block was voltage dependent, increasing with depolarization, indicating that block was coupled to the electric field of the membrane. Proton block was not described by the Woodhull equation for noncompetitive voltage-dependent block, but was described by an equation for cooperative competitive inhibition that included voltage-dependent block from the Woodhull equation. Proton block was still present after replacing the eight negative charges in the ring of charge at the entrance to the intracellular vestibule by uncharged amino acids. Thus, the ring of charge is not the site of proton block or of competitive inhibition of K(+)(i) action by H(+)(i). With 150 mM symmetrical KCl, unitary current amplitudes increased with depolarization, reaching 66 pA at +350 mV (pH(i) 7.0). The increase in amplitude with voltage became sublinear for voltages >100 mV. The sublinearity was unaffected by removing from the intracellular solutions Ca(2+) and Ba(2+) ions, the Ca(2+) buffers EGTA and HEDTA, the pH buffer TES, or by replacing Cl(-) with MeSO(3)(-). Proton block accounted for approximately 40% of the sublinearity at +200 mV and pH 7.0, indicating that factors in addition to proton block contribute to the sublinearity of the unitary currents through BK channels. 相似文献
13.
The effects of pH of the external medium on amplitude of currents through single sodium channels at the membrane of cultured neuroblastoma cells were investigated in mice belonging to strain C 1300, clone N18A-1. Currents through single sodium channels in isolated membrane segments (outside-out configuration) were registered with normal (7.2) and reduced (5.4) pH levels in the external medium. Reducing the pH to 5.4 was found to decrease current amplitude reversibly by about twofold (–10 to –30 mV for test potentials). Findings would confirm that the depression of macroscopic sodium currents produced by reducing the pH of the extracellular solution is due to a decline in ionic flow through single open sodium channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 101–105, January–February, 1989. 相似文献
14.
Cherny VV Murphy R Sokolov V Levis RA DeCoursey TE 《The Journal of general physiology》2003,121(6):615-628
Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi < or = 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, sigmaH2. We analyze sigmaH2 data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi < or = 5.5 near Vthreshold (the voltage at which gH turns on), single channel-like current events were observed with amplitudes 7-16 fA. 相似文献
15.
Attenuation of proton currents by methanol in a dioxolane-linked gramicidin A channel in different lipid bilayers.
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The mobility of protons in a dioxolane-linked gramicidin A channel (D1) is comparable to the mobility of protons in aqueous solutions (Cukierman, S., E. P. Quigley, and D. S. Crumrine. 1997. Biophys. J. 73:2489-2502). Aliphatic alcohols decrease the mobility of H+ in aqueous solutions. In this study, the effects of methanol on proton conduction through D1 channels were investigated in different lipid bilayers and at different HCl concentrations. Methanol attenuated H+ currents in a voltage-independent manner. Attenuation of proton currents was also independent of H+ concentrations in solution. In phospholipid bilayers, methanol decreased the single channel conductance to protons without affecting the binding affinity of protons to bilayers. In glycerylmonooleate membranes, the attenuation of single channel proton conductances qualitatively resembled the decrease of conductivities of HCl solutions by methanol. However, in both types of lipid bilayers, single channel proton conductances through D1 channels were considerably more attenuated than the conductivities of different HCl solutions. This suggests that methanol modulates single proton currents through D1 channels. It is proposed that, on average, one methanol molecule binds to a D1 channel, and attenuates H+ conductance. The Gibbs free energy of this process (DeltaG0) is approximately 1.2 kcal/mol, which is comparable to the free energy of decrease of HCl conductivity in methanol solutions (1.6 kcal/mol). Apolar substances like urea and glucose that do not transport protons in HCl solutions and do not permeate D1 channels decreased solution conductivity and single channel conductance by a considerably larger proportion than methanol. Cs+ currents through D1 channels were considerably less (fivefold) attenuated by methanol than proton currents. It is proposed that methanol partitions inside the pore of gramicidin channels and delays the transfer of protons between water and methanol molecules, causing a significant attenuation of the single channel proton conductance. Gramicidin channels offer an interesting experimental model to study proton hopping along a single chain of water molecules interrupted by a single methanol molecule. 相似文献
16.
Boris Musset Susan ME Smith Sindhu Rajan Vladimir V Cherny Deri Morgan Thomas E DeCoursey 《Channels (Austin, Tex.)》2010,4(4):260-265
The voltage-gated proton channel exists as a dimer, although each protomer has a separate conduction pathway, and when forced to exist as a monomer, most major functions are retained. However, the proton channel protomers appear to interact during gating. Proton channel dimerization is thought to result mainly from coiled-coil interaction of the intracellular C-termini. Several types of evidence are discussed that suggest that the dimer conformation may not be static, but is dynamic and can sample different orientations. Zn2+ appears to link the protomers in an orientation from which the channel(s) cannot open. A tandem WT-WT dimer exhibits signs of cooperative gating, indicating that despite the abnormal linkage, the correct orientation for opening can occur. We propose that C-terminal interaction functions mainly to tether the protomers together. Comparison of the properties of monomeric and dimeric proton channels speaks against the hypothesis that enhanced gating reflects monomer-dimer interconversion.Key words: voltage-gated proton channels, voltage gating, voltage-sensing domains, phagocytes, coiled-coil, oligomerization, proton currents, pH, dimerization, C-terminus 相似文献
17.
Tianbo Li Youshan Yang Cecilia M. Canessa 《The Journal of biological chemistry》2010,285(29):22706-22712
Acid-sensing ion channels (ASICs) are proton-activated channels expressed in neurons of the central and peripheral nervous systems where they modulate neuronal activity in response to external increases in proton concentration. The size of ASIC1 currents evoked by a given local acidification is determined by the number of channels in the plasma membrane and by the apparent proton affinities for activation and steady-state desensitization of the channel. Thus, the magnitude of the pH drop and the value of the baseline pH both are functionally important. Recent characterization of ASIC1s from an increasing number of species has made evident that proton affinities of these channels vary across vertebrates. We found that in species with high baseline plasma pH, e.g. frog, shark, and fish, ASIC1 has high proton affinity compared with the mammalian channel. The β1-β2 linker in the extracellular domain, specifically by the substitution M85L, determines the interspecies differences in proton affinities and also the time course of ASIC1 macroscopic currents. The mechanism underlying these observations is a delay in channel opening after application of protons, most likely by stabilizing a closed conformation that decreases the apparent affinity to protons and also slows the rise and decay phases of the current. Together, the results suggest evolutionary adaptation of ASIC1 to match the value of the species-specific plasma pH. At the molecular level, adaptation is achieved by substitutions of nonionizable residues rather than by modification of the channel proton sensor. 相似文献
18.
Chilman Bae Frederick Sachs Philip A. Gottlieb 《The Journal of biological chemistry》2015,290(8):5167-5173
PIEZO1 is a recently cloned eukaryotic cation-selective channel that opens with mechanical force. We found that extracellular protonation inhibits channel activation by ≈90% by increased occupancy in the closed or the inactivated state. Titration between pH 6.3 and 8.3 exhibited a pK of ≈6.9. The steepness of the titration data suggests positive cooperativity, implying the involvement of at least two protonation sites. Whole-cell recordings yielded results similar to patches, and pH 6.5 reduced whole-cell currents by >80%. The effects were reversible. To assess whether pH acts on the open or the inactivated state, we tested a double-mutant PIEZO1 that does not inactivate. Cell-attached patches and whole-cell currents from this mutant channel were pH-insensitive. Thus, protonation appears to be associated with domain(s) of the channel involved with inactivation. pH also did not affect mutant channels with point mutations at position 2456 that are known to exhibit slow inactivation. To determine whether the physical properties of the membrane are altered by pH and thereby affect channel gating, we measured patch capacitance during mechanical stimuli at pH 6.5 and 7.3. The rate constants for changes in patch capacitance were independent of pH, suggesting that bilayer mechanics are not involved. In summary, low pH stabilizes the inactivated state. This effect may be important when channels are activated under pathological conditions in which the pH is reduced, such as during ischemia. 相似文献
19.
Rat P2X2 receptors open at an undetectably low rate in the absence of ATP. Furthermore, two allosteric modulators, zinc and acidic pH, cannot by themselves open these channels. We describe here the properties of a mutant receptor, K69C, before and after treatment with the thiol-reactive fluorophore Alexa Fluor 546 C5-maleimide (AM546). Xenopus oocytes expressing unmodified K69C were not activated under basal conditions nor by 1,000 µM ATP. AM546 treatment caused a small increase in the inward holding current which persisted on washout and control experiments demonstrated this current was due to ATP independent opening of the channels. Following AM546 treatment, zinc (100 µM) or acidic external solution (pH 6.5) elicited inward currents when applied without any exogenous ATP. In the double mutant K69C/H319K, zinc elicited much larger inward currents, while acidic pH generated outward currents. Suramin, which is an antagonist of wild type receptors, behaved as an agonist at AM546-treated K69C receptors. Several other cysteine-reactive fluorophores tested on K69C did not cause these changes. These modified receptors show promise as a tool for studying the mechanisms of P2X receptor activation. 相似文献
20.
Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions 总被引:12,自引:8,他引:4
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We studied the pH dependence of the proton-induced current fluctuations that appear in single open L-type Ca channels when monovalent ions are the charge carriers. We used different methods of analysis to obtain kinetic measurements even under conditions where the individual transitions were too fast to be resolved directly as discrete current steps between two conductance levels. The reciprocal of the dwell times at the high conductance level increased linearly with the pipette proton activity, with a slope that was similar for Cs, K, and Na as permeant ions. Contrary to the expectation for a simple model in which the high and low conductances represent the unprotonated and protonated states of the channel, respectively, the dwell times at the low conductance level were also pH dependent and lengthened with increasing proton activity. At all pH values the dwell times at the low conductance level were longest with Cs as permeant ion and shortened in the order Cs greater than K greater than Na. We introduce a more general model of the protonation cycle in which the channel is represented by four states and can be protonated and deprotonated both at the high and low conductance levels. The conductance change is represented by a conformational change of the channel protein. We discuss the validity of this model and its implications for the mechanism by which protons interact with ion permeation through L-type Ca channels. 相似文献