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1.
《The Journal of general physiology》1994,103(2):249-278
Kinetics of single voltage-dependent Shaker potassium channels expressed in Xenopus oocytes were studied in the absence of fast N-type inactivation. Comparison of the single-channel first latency distribution and the time course of the ensemble average current showed that the activation time course and its voltage dependence are largely determined by the transitions before first opening. The open dwell time data are consistent with a single kinetically distinguishable open state. Once the channel opens, it can enter at least two closed states which are not traversed frequently during the activation process. The rate constants for the transitions among these closed states and the open state are nearly voltage-independent at depolarized voltages (> - 30 mV). During the deactivation process at more negative voltages, the channel can close directly to a closed state in the activation pathway in a voltage-dependent fashion. 相似文献
2.
《The Journal of general physiology》1994,103(2):321-362
Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage. 相似文献
3.
Recently, the structure of the Shaker channel Kv1.2 has been determined at a 2.9-angstroms resolution. This opens new possibilities in deciphering the mechanism underlying the function of voltage-gated potassium (Kv) channels. Molecular dynamics simulations of the channel, embedded in a membrane environment show that the channel is in its open state and that the gating charges carried by S4 are exposed to the solvent. The hydrated environment of S4 favors a local collapse of the electrostatic potential, which generates high electric-field gradients around the arginine gating charges. Comparison to experiments suggests furthermore that activation of the channel requires mainly a lateral displacement of S4. Overall, the results agree with the transporter model devised for Kv channels from electrophysiology experiments, and provide a possible pathway for the mechanistic response to membrane depolarization. 相似文献
4.
C Y Lee 《FEBS letters》1992,311(2):81-84
This paper proposes a detailed gating mechanism for the N-methyl-D-aspartate (NMDA) channel. In the NMDAR1 subunit, the signal of agonist binding may be carried from Y456 to W590 through an electron transport chain, including W480 which could be the glycine modulatory site. The channel's opening may arise from repulsion between negatively charged W590s, analogous to W435s of the Shaker K+ channel. The cyclic nucleotide-gated channels may be activated by a similar mechanism, but the opening of nicotinic acetylcholine receptor (nAChR) channels is likely to be initiated by the formation of tyrosine radicals. The role of disulfide-bonded cysteines in the redox modulation can also be explained. 相似文献
5.
Based on the structure of the KcsA potassium channel, the Shaker K+ channel is thought to have, near the middle of the membrane, a cavity that can be occupied by a permeant or a blocking cation. We have studied the interaction between cations in the cavity and the activation gate of the channel, using a set of monovalent cations together with Shaker mutants that modify the structure of the cavity. Our results show that reducing the size of the side chain at position 470 makes it possible for the mutant channel, unlike native Shaker, to close with tetraethylammonium (TEA+) or the long-chain TEA-derivative C10+ trapped inside the channel. Neither I470 mutants nor Shaker can close when N-methyl-glucamine (NMG+) is in the channel, even though this ion is smaller than C10+. Apparently, the carbohydrate side chain of NMG+ prevents gate closing. Gating currents recorded from Shaker and I470C were measured in the presence of different intracellular cations to further analyze the interaction of cations with the gate. Our results suggest that the cavity in Shaker is so small that even permeant cations like Rb+ or Cs+ must leave the cavity before the channel gate can close. 相似文献
6.
Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. 总被引:6,自引:0,他引:6
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Steady-state and kinetic properties of gating currents of the Shaker K+ channels were studied in channels expressed in Xenopus oocytes and recorded with the cut-open oocyte voltage clamp. The charge versus potential (Q-V) curve reveals at least two components of charge, the first moving in the hyperpolarized region (V1/2 = -63 mV) and the second, with a larger apparent valence, moving in the more depolarized region (V1/2 = -44 mV). The kinetic analysis of gating currents revealed also two exponential decaying components that corresponded in their voltage dependence with the charge components described in the steady-state. The first component was found to correlate with the effects of prepulses that produce the Cole-Moore shift of the ionic and gating currents and seems to be occurring completely within closed conformations of the channel. The second component seems to be related to the events occurring between the closed states just preceding, but not including, the transition to the open state. The ON and OFF gating currents exhibit a pronounced rising phase at potentials at which the second component becomes important, and this region corresponds to the potential range where the channel opens. The results could not be explained with simple parallel models, but the data can be fitted to a sequential model that could be related to a first rearrangement of the putative four subunits in cooperative fashion, followed by a concerted charge movement that leads to the open channel. The first series of charge movements are produced by transitions between several closed states carrying less than two electronic charges per step, while a step carrying about 3.5 electronic charges can explain the second component. This step is followed by the transition to the open state carrying less than 0.5 electronic charges. This model is able to reproduce all the kinetic and steady-state properties of the gating currents and predicts many of the properties of the ionic currents. 相似文献
7.
To investigate of the gating properties in the voltage-activated potassium channel, we have mutated a variety of S2 and S4 residues in the Shaker potassium protein. Results showed that the R365C and R368C, but not the E283C, R362C, R365S, R368S or the ShB-IR, were sensitive to micromolar concentrations of Cd(2+) ions. This indicates that R365 and R368 play a crucial role in the channel gating due to a conformational modulation of the channel structure. Doubly mutated channels of the E283C/R365E and E283C/R368E caused a transient increase in current amplitude, which reached a peak within a few seconds and then decreased toward initial levels, despite the continual presence of Cd(2+). Taken together, our results suggest that E283, R365, and R368 form a network of strong, local, and electrostatic interactions that relate closely to the mechanism of the channel gating. 相似文献
8.
Various experiments have suggested that the S4 segment in voltage-dependent Na(+) and K(+) channels is in contact with a solvent-accessible cavity. We explore the consequences of the existence of such a cavity through the electrostatic effects on the gating currents of Shaker K(+) channels under conditions of reduced ionic strength S. We observe that approximately 10-fold reductions of intracellular S produce reductions of the measured gating charge of approximately 10%. These effects continue at even lower values of S. The reduction of gating charge when S is reduced by 10-fold at the extracellular surface is much smaller (approximately 2%). Shifts of the Q(V) curve because of a reduced S are small (<10 mV in size), which is consistent with very little fixed surface charge. Continuum electrostatic calculations show that the S effects on gating charge can be explained by the alteration of the local potential in an intracellular conical cavity of 20-24-A depth and 12-A aperture, and a smaller extracellular cavity of 3-A depth and the same aperture. In this case, the attenuation of the membrane potential at low S leads to reduction of the apparent gating charge. We suggest that this cavity is made by a bundle of transmembrane helices, and that the gating charge movement occurs by translocation of charged residues across a thin septum of approximately 3-7 A thickness. 相似文献
9.
External Ba2+ speeds the OFF gating currents (IgOFF) of Shaker K+ channels but only upon repolarization from potentials that are expected to open the channel pore. To study this effect we used a nonconducting and noninactivating mutant of the Shaker K+ channel, ShH4-IR (W434F). External Ba2+ slightly decreases the quantity of ON gating charge (QON) upon depolarization to potentials near -30 mV but has little effect on the quantity of charge upon stepping to more hyperpolarized or depolarized potentials. More strikingly, Ba2+ significantly increases the decay rate of IgOFF upon repolarization to -90 mV from potentials positive to approximately -55 mV. For Ba2+ to have this effect, the depolarizing command must be maintained for a duration that is dependent on the depolarizing potential (> 4 ms at -30 mV and > 1 ms at 0 mV). The actions of Ba2+ on the gating current are dose-dependent (EC50 approximately 0.2 mM) and are not produced by either Ca2+ or Mg2+ (2 mM). The results suggest that Ba2+ binds to a specific site on the Shaker K+ channel that destabilizes the open conformation and thus facilitates the return of gating charge upon repolarization. 相似文献
10.
Voltage-dependent gating of Shaker A-type potassium channels in Drosophila muscle 总被引:26,自引:6,他引:20
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The voltage-dependent gating mechanism of A1-type potassium channels coded for by the Shaker locus of Drosophila was studied using macroscopic and single-channel recording techniques on embryonic myotubes in primary culture. From a kinetic analysis of data from single A1 channels, we have concluded that all of the molecular transitions after first opening, including the inactivation transition, are voltage independent and therefore not associated with charge movement through the membrane. In contrast, at least some of the activation transitions leading to first opening are considerably voltage dependent and account for all of the voltage dependence seen in the macroscopic currents. This mechanism is similar in many ways to that of vertebrate neuronal voltage-sensitive sodium channels, and together with the sequence similarities in the S4 region suggests a conserved mechanism for voltage-dependent gating among channels with different selectivities. By testing independent and coupled models for activation and inactivation we have determined that the final opening transition and inactivation are not likely to arise from the independent action of multiple subunits, each with simple gating transitions, but rather come about through their aggregate properties. A partially coupled model accurately reproduces all of the single-channel and macroscopic data. This model will provide a framework on which to organize and understand alterations in gating that occur in Shaker variants and mutants. 相似文献
11.
Glycosylation affects the rate of traffic of the Shaker potassium channel through the secretory pathway 总被引:2,自引:0,他引:2
We have examined the effect of glycosylation on the traffic of the voltage-gated Shaker potassium channel through the secretory pathway of mammalian cells. Shaker is glycosylated on two asparagines (N259 and N263) in the first extracellular loop. Electrophysiological experiments indicate that glycosylation is not necessary for channel integrity [Santacruz-Toloza et al. (1994) Biochemistry 33, 5607]. Consistent with this, we observe that unglycosylated N259Q+N263Q mutant channel forms oligomers as efficiently as the wild type and that this occurs in the endoplasmic reticulum. We have compared the kinetics of secretory traffic of the wild-type glycosylated and the N259Q+N263Q unglycosylated channels. Surface biotinylation of newly synthesized proteins indicates that the rate of delivery of the unglycosylated channel to the cell surface is slower than that of wild type. We have further dissected channel traffic using quantitative imaging. We observe that mutant channel traffics more slowly from the endoplasmic reticulum to the Golgi than wild type at 20 degrees C. This may contribute to the slowed delivery of the mutant to the cell surface. Neither the surface fraction at steady state nor the stability of Shaker is significantly affected by glycosylation in COS cells. 相似文献
12.
The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523-526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating. 相似文献
13.
The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1. 相似文献
14.
In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521-532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels. 相似文献
15.
Binding of a gating modifier toxin induces intersubunit cooperativity early in the Shaker K channel's activation pathway
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Potassium currents from voltage-gated Shaker K channels activate with a sigmoid rise. The degree of sigmoidicity in channel opening kinetics confirms that each subunit of the homotetrameric Shaker channel undergoes more than one conformational change before the channel opens. We have examined effects of two externally applied gating modifiers that reduce the sigmoidicity of channel opening. A toxin from gastropod mucus, 6-bromo-2-mercaptotryptamine (BrMT), and divalent zinc are both found to slow the same conformational changes early in Shaker's activation pathway. Sigmoidicity measurements suggest that zinc slows a conformational change independently in each channel subunit. Analysis of activation in BrMT reveals cooperativity among subunits during these same early steps. A lack of competition with either agitoxin or tetraethylammonium indicates that BrMT binds channel subunits outside of the external pore region in an allosterically cooperative fashion. Simulations including negatively cooperative BrMT binding account for its ability to induce gating cooperativity during activation. We conclude that cooperativity among K channel subunits can be greatly altered by experimental conditions. 相似文献
16.
Ping Li Zhuxi Chen Haiyan Xu Haifeng Sun Hao Li Hong Liu Huaiyu Yang Zhaobing Gao Hualiang Jiang Min Li 《Cell research》2013,23(9):1106-1118
Voltage-gated potassium (Kv) channels derive their voltage sensitivity from movement of gating charges in voltage-sensor domains (VSDs). The gating charges translocate through a physical pathway in the VSD to open or close the channel. Previous studies showed that the gating charge pathways of Shaker and Kv1.2-2.1 chimeric channels are occluded, forming the structural basis for the focused electric field and gating charge transfer center. Here, we show that the gating charge pathway of the voltage-gated KCNQ2 potassium channel, activity reduction of which causes epilepsy, can accommodate various small molecule ligands. Combining mutagenesis, molecular simulation and electrophysiological recording, a binding model for the probe activator, ztz240, in the gating charge pathway was defined. This information was used to establish a docking-based virtual screening assay targeting the defined ligand-binding pocket. Nine activators with five new chemotypes were identified, and in vivo experiments showed that three ligands binding to the gating charge pathway exhibit significant anti-epilepsy activity. Identification of various novel activators by virtual screening targeting the pocket supports the presence of a ligand-binding site in the gating charge pathway. The capability of the gating charge pathway to accommodate small molecule ligands offers new insights into the gating charge pathway of the therapeutically relevant KCNQ2 channel. 相似文献
17.
Kargol A Hosein-Sooklal A Constantin L Przestalski M 《General physiology and biophysics》2004,23(1):53-75
Nonequilibrium response spectroscopy (NRS) has been proposed recently to complement standard electrophysiological techniques used to investigate ion channels. It involves application of rapidly oscillating potentials that drive the ion channel ensemble far from equilibrium. It is argued that new, so far undiscovered features of ion channel gating kinetics may become apparent under such nonequilibrium conditions. In this paper we explore the possibility of using regular, sinusoidal voltages with the NRS protocols to facilitate Markov model selection for ion channels. As a test case we consider the Shaker potassium channel for which various Markov models have been proposed recently. We concentrate on certain classes of such models and show that while some models might be virtually indistinguishable using standard methods, they show marked differences when driven with an oscillating voltage. Model currents are compared to experimental data obtained for the Shaker K+ channel expressed in mammalian cells (tsA 201). 相似文献
18.
A physical model of potassium channel activation: from energy landscape to gating kinetics
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We have developed a method for rapidly computing gating currents from a multiparticle ion channel model. Our approach is appropriate for energy landscapes that can be characterized by a network of well-defined activation pathways with barriers. To illustrate, we represented the gating apparatus of a channel subunit by an interacting pair of charged gating particles. Each particle underwent spatial diffusion along a bistable potential of mean force, with electrostatic forces coupling the two trajectories. After a step in membrane potential, relaxation of the smaller barrier charge led to a time-dependent reduction in the activation barrier of the principal gate charge. The resulting gating current exhibited a rising phase similar to that measured in voltage-dependent ion channels. Reduction of the two-dimensional diffusion landscape to a circular Markov model with four states accurately preserved the time course of gating currents on the slow timescale. A composite system containing four subunits leading to a concerted opening transition was used to fit a series of gating currents from the Shaker potassium channel. We end with a critique of the model with regard to current views on potassium channel structure. 相似文献
19.
Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions. 相似文献
20.
Shaker K+ channels were expressed in outside-out macropatches excised from Xenopus oocytes, and the effects on gating of removal of extracellular Ca2+ were examined in the complete absence of intracellular divalent cations. Removal of extracellular Ca2+ by perfusion with EDTA-containing solution caused a small negative shift in the channel's voltage-activation curve and led to an increased nonselective leak, but did not otherwise alter or disrupt the channels. The results contradict the proposal that Ca2+ is an essential component required for maintenance of ion selectivity and proper gating of Kv-type K+ channels. The large nonselective leak in Ca2+-free conditions was found to be a patch-seal phenomenon related to F- ion in the recording pipette. 相似文献