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Asipu Sivaprasadarao - One of the best experts on this subject based on the ideXlab platform.
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Role of hydrophobic and ionic forces in the movement of S4 of the Shaker Potassium Channel.
Molecular membrane biology, 2012Co-Authors: David J. S. Elliott, Edward J. Neale, Tim S. Munsey, John P. Bannister, Asipu SivaprasadaraoAbstract:AbstractVoltage-gated ion (K+, Na+, Ca2+) Channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion Channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on Channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker Potassium Channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating ...
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Evidence for Intersubunit Interactions between S4 and S5 Transmembrane Segments of the Shaker Potassium Channel
The Journal of biological chemistry, 2003Co-Authors: Edward J. Neale, David J. S. Elliott, Malcolm Hunter, Asipu SivaprasadaraoAbstract:Voltage-gated Potassium Channels are transmembrane proteins made up of four subunits, each comprising six transmembrane (S1-S6) segments. S1-S4 form the voltage-sensing domain and S5-S6 the pore domain with its central pore. The sensor domain detects membrane depolarization and transmits the signal to the activation gates situated in the pore domain, thereby leading to Channel opening. An understanding of the mechanism by which the sensor communicates the signal to the pore requires knowledge of the structure of the interface between the voltage-sensing and pore domains. Toward this end, we have introduced single cysteine mutations into the extracellular end of S4 (positions 356 and 357) in conjunction with a cysteine in S5 (position 418) of the Shaker Channel and expressed the mutants in Xenopus oocytes. We then examined the propensity of each pair of engineered cysteines to form a metal bridge or a disulfide bridge, respectively, by examining the effect of Cd2+ ions and copper phenanthroline on the K+ conductance of a whole oocyte. Both reagents reduced currents through the S357C,E418C double mutant Channel, presumably by restricting the movements necessary for coupling the voltage-sensing function to pore opening. This inhibitory effect was seen in the closed state of the Channel and with heteromers composed of S357C and E418C single mutant subunits; no effect was seen with homomers of any of the single mutant Channels. These data indicate that the extracellular end of S4 lies in close proximity to the extracellular end of the S5 of the neighboring subunit in closed Channels.
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Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker Potassium Channel.
The Journal of biological chemistry, 2002Co-Authors: Qadeer Aziz, Tim S. Munsey, Christopher J. Partridge, Asipu SivaprasadaraoAbstract:Abstract Voltage-gated Potassium (Kv) Channels are integral membrane proteins, composed of four subunits, each comprising six (S1–S6) transmembrane segments. S1–S4 comprise the voltage-sensing domain, and S5–S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the Channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K Channel and expressed the mutant Channel inXenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant Channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during Channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the Channel. The latter two effects suggest stabilization of the open state of the Channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K Channel is reduced.
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Effect of cysteine substitutions on the topology of the S4 segment of the Shaker Potassium Channel: implications for molecular models of gating.
The Journal of Physiology, 1999Co-Authors: M H Wang, David J. S. Elliott, S P Yusaf, D Wray, Asipu SivaprasadaraoAbstract:The gating properties of voltage-gated Potassium Channels are largely determined by the amino acid sequence of their S4 segments. To investigate the nature of S4 movement during gating, we introduced single cysteines into the S4 segment of the Shaker Potassium Channel and expressed the mutants in Xenopus oocytes. We then measured the conductance-voltage (g-V) relationships and the rate and the voltage dependence of movement of the engineered cysteines, using p-chloromercuribenzene sulphonate (pCMBS) as a probe. Mutation of charged residues at positions 362, 365 and 368, but not the uncharged residues (positions 360, 361, 363, 364 and 366), to cysteines shifted the g-V relationships to more positive potentials. Mutant Channels in which cysteines replaced the charged residues at positions 362 and 365 (R362C and R365C) reacted faster with pCMBS than those in which cysteines were introduced in place of uncharged residues at positions 360 and 361 (I360C and L361C). Furthermore, the R365C mutant Channel reacted with pCMBS even at hyperpolarised (-120 mV) potentials. Currents expressed by the doubly mutated R365S/V367C and R368S/V367C Channels, but not the singly mutated V367C Channel, were inhibited by pCMBS. Moreover, the R368C mutant Channel was also affected by pCMBS. Voltage dependence of block by pCMBS (2 min exposure) was steeper for L366C than for L361C and V363C mutant Channels (effective charge 2.19, 1.41 and 1.45, respectively). The voltage dependence of the pCMBS effect was also shifted to more depolarising potentials the deeper in the membrane the position of the residue mutated to cysteine (voltages for half-maximal effect -107, -94 and -73 mV for positions 361, 363 and 366, respectively). Our data show firstly that charge-neutralising mutations in S4 alter the topology of this region such that the membrane-spanning portion of S4 is reduced. Secondly, our data for the other mutant Channels suggest that S4 might move in at least two sequential steps, and can move up to its maximal limit even at the resting potential of the cell. Voltage-gated Potassium Channels comprise a large group of integral membrane proteins, members of which are present in almost all cell types (Hille, 1992; Jan & Jan, 1997). They contain two functional elements, a voltage sensor with which they sense voltage changes across the cell membrane and a selective pore through which K+ ions permeate (Sigworth, 1994; Jan & Jan, 1997; Yellen, 1998). These functional elements are coupled in such a way that when the membrane is depolarised the sensor detects the change and transmits the signal to the pore which then opens to let Potassium ions flow down the electrochemical gradient. Despite numerous studies (Papazian et al. 1991, 1995; Seoh et al. 1996; Larsson et al. 1996; Yusaf et al. 1996; Cha & Bezanilla, 1997; Starace et al. 1997; Baker et al. 1998) the molecular details of this process are still unclear. One reason for this is the lack of three-dimensional structural information for these proteins. Recent X-ray diffraction data (Doyle et al. 1998) from crystals of KcsA, a bacterial K+ Channel, have revealed the structure of the pore and have provided an insight into the mechanism by which K+ Channels select K+ ions over other cations. However, KcsA, unlike voltage-gated K+ Channels, lacks the voltage sensor. For this reason, although the molecular architecture of the pore is now known, the structure of the voltage sensor and the molecular mechanism by which it controls Channel opening remain unclear. In the absence of direct structural data, site-directed mutagenesis, in conjunction with biochemical (Tiwari-Woodruff et al. 1997) and electrophysiological (Aggarwal & MacKinnon, 1996; Larsson et al. 1996; Yusaf et al. 1996; Cha & Bezanilla, 1997; Baker et al. 1998) approaches, has been used to investigate the structure of the sensor and the mechanisms by which it controls Channel gating. The data suggest that the putative transmembrane segments S2, S3 and S4 together constitute the voltage sensor of the Channel. These segments contain highly conserved charged residues: the S4 segment contains four to seven positively charged residues (arginine or lysine) separated from one another by two hydrophobic residues and the S2 and S3 segments contain negatively charged residues (glutamic or aspartic acid) at conserved positions. Positive charges in S4 are thought to form salt bridges with the negative charges in the S2 and S3 segments and seem to make a major contribution to the gating charge of the Channel (Tiwari-Woodruff et al. 1997). Some of these charges sense voltage changes across the membrane and move across the transmembrane field. Recent experiments have provided evidence that during depolarisation the S4 segment moves towards the extracellular phase, carrying these gating charges through the transmembrane field (Larsson et al. 1996; Yusaf et al. 1996; Baker et al. 1998). A large number of voltage-gated K+ Channels have been cloned (Jan & Jan, 1997); they differ from one another with respect to the voltage at which they begin to activate (threshold of activation) and the range of voltage (slope) over which they become fully activated. Changes in these properties can be readily introduced into a Channel by mutating residues that contribute to the voltage sensing property of the Channel. This has provided the opportunity to examine the relationship between the gating properties and the kinetics of S4 movement using mutants of cloned Channels. Accordingly, we have engineered mutations into the S4 segment of the Shaker Channel, expressed them in Xenopus oocytes and, using substituted cysteine accessibility mutagenesis (SCAM) in conjunction with two-electrode voltage clamp, examined the effect of these mutations on S4 movement. Our studies show that: (i) neutralisation of charged residues by replacement with uncharged residues, which shifts the conductance-voltage relationships to the right, alters the transmembrane topology and spatial organisation of the S4 segment, (ii) S4 might move in at least two sequential steps and (iii) S4 movement is highly dynamic, with movements occurring even at the resting potential of the cell. The implications of these new data for the current molecular models of gating are discussed.
Fred J. Sigworth - One of the best experts on this subject based on the ideXlab platform.
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Can Shaker Potassium Channels be Locked in the Deactivated State
The Journal of general physiology, 2004Co-Authors: Youshan Yang, Yangyang Yan, Fred J. SigworthAbstract:For structural studies it would be useful to constrain the voltage sensor of a voltage-gated Channel in its deactivated state. Here we consider one Shaker Potassium Channel mutant and speculate about others that might allow the Channel to remain deactivated at zero membrane potential. Ionic and gating currents of F370C Shaker, expressed in Xenopus oocytes, were recorded in patches with internal application of the methanethiosulfonate reagent MTSET. It appears that the voltage dependence of voltage sensor movement is strongly shifted by reaction with internal MTSET, such that the voltage sensors appear to remain deactivated even at positive potentials. A disadvantage of this construct is that the rate of modification of voltage sensors by MTSET is quite low, ∼0.17 mM−1·s−1 at −80 mV, and is expected to be much lower at depolarized potentials.
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Potassium Channel mechanics.
Neuron, 2001Co-Authors: Fred J. SigworthAbstract:What is the moving part that switches an ion Channel's current on and off? In this issue of Neuron del Camino and Yellen (2001) exploit scanning cysteine mutagenesis and sulfhydryl reagents to show that the intracellular end of the S6 helices forms a mechanical gate for the Shaker Potassium Channel.
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Activation of Shaker Potassium Channels. III. An activation gating model for wild-type and V2 mutant Channels.
The Journal of general physiology, 1998Co-Authors: Nathan E. Schoppa, Fred J. SigworthAbstract:A functional kinetic model is developed to describe the activation gating process of the Shaker Potassium Channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single Channel ionic currents. These data were obtained from the normally activating wild-type Channel as well as a mutant Channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker 9s symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the Channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the Channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e 0 ), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e 0 ). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of Channel opening at Shaker 9s activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.
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Role of the S3-S4 Linker in Shaker Potassium Channel Activation
The Journal of general physiology, 1997Co-Authors: Rajesh Mathur, Jie Zheng, Yangyang Yan, Fred J. SigworthAbstract:Structural models of voltage-gated Channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker Channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7- or 9-residue sequences from Shab, Shaw, or Shal. As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw Channels resulted in a three- to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during Channel activation. The linker, which in some Channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in Potassium Channels, as previously has been suggested in the case of calcium Channels.
Richard W. Aldrich - One of the best experts on this subject based on the ideXlab platform.
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A Mutation in S6 of Shaker Potassium Channels Decreases the K+ Affinity of an Ion Binding Site Revealing Ion–Ion Interactions in the Pore
The Journal of general physiology, 1998Co-Authors: Eva M. Ogielska, Richard W. AldrichAbstract:Under physiological conditions, Potassium Channels are extraordinarily selective for Potassium over other ions. However, in the absence of Potassium, certain Potassium Channels can conduct sodium. Sodium flux is blocked by the addition of low concentrations of Potassium. Potassium affinity, and therefore the ability to block sodium current, varies among Potassium Channel subtypes (Korn, S.J., and S.R. Ikeda. 1995. Science. 269:410-412; Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539-550). The Shaker Potassium Channel conducts sodium poorly in the presence of very low (micromolar) Potassium due to its high Potassium affinity (Starkus, J.G., L. Kuschel, M.D. Rayner, and S.H. Heinemann. 1997. J. Gen. Physiol. 110:539-550; Ogielska, E.M., and R. W. Aldrich. 1997. Biophys. J. 72:A233 [Abstr.]). We show that changing a single residue in S6, A463C, decreases the apparent internal Potassium affinity of the Shaker Channel pore from the micromolar to the millimolar range, as determined from the ability of Potassium to block the sodium currents. Independent evidence that A463C decreases the apparent affinity of a binding site in the pore comes from a study of barium block of Potassium currents. The A463C mutation decreases the internal barium affinity of the Channel, as expected if barium blocks current by binding to a Potassium site in the pore. The decrease in the apparent Potassium affinity in A463C Channels allows further study of possible ion interactions in the pore. Our results indicate that sodium and Potassium can occupy the pore simultaneously and that multiple occupancy results in interactions between ions in the Channel pore.
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Shaker Potassium Channel gating. II: Transitions in the activation pathway.
The Journal of general physiology, 1994Co-Authors: William N Zagotta, Toshinori Hoshi, Jeremy S. Dittman, Richard W. AldrichAbstract:Voltage-dependent gating behavior of Shaker Potassium Channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-Channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.
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Shaker Potassium Channel gating. I: Transitions near the open state.
The Journal of general physiology, 1994Co-Authors: Toshinori Hoshi, William N Zagotta, Richard W. AldrichAbstract: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.
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Shaker Potassium Channel gating. III: Evaluation of kinetic models for activation
The Journal of general physiology, 1994Co-Authors: William N Zagotta, Toshinori Hoshi, Richard W. AldrichAbstract: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.
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Functional stoichiometry of Shaker Potassium Channel inactivation
Science (New York N.Y.), 1993Co-Authors: Roderick Mackinnon, Richard W. Aldrich, Alice W. LeeAbstract:Shaker Potassium Channels from Drosophila are composed of four identical subunits. The contribution of a single subunit to the inactivation gating transition was investigated. Channels carrying a specific mutation in a single subunit can be labeled in a heterogeneous population and studied quantitatively with scorpion toxin sensitivity as a selection tag. Linkage within a single subunit of a mutation that removes the inactivation gate to a second mutation that affects scorpion toxin sensitivity demonstrates that only a single gate is necessary to produce inactivation. The inactivation rate constant for Channels with a single gate was one-fourth that of Channels with four gates. In contrast, the rate of recovery from inactivation was independent of the number of gates. It appears that each of the four open inactivation gates in a Shaker Potassium Channel is independent, but only one of the four gates closes in a mutually exclusive manner.
Chanda - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Mapping of Interactions in the Voltage-Sensor Pore Interface of the Shaker Potassium Channel
Biophysical Journal, 2015Co-Authors: Kevin Oelstrom, Ana I. Fernández-mariño, ChandaAbstract:Changes in the membrane potential of an excitable cell induce movement of voltage-sensing units within voltage-gated ion Channels (VGICs). The energy associated with this movement is transmitted to the Channel gate, ultimately promoting its opening. Numerous proposals have been made as to how this signal is relayed from the voltage-sensors to the Channel gate, such as side chain interactions and backbone movement; however, it has been difficult to ascertain the precise molecular mechanisms underlying energy transduction. Recently, it was shown that median voltage estimates from the charge-voltage relationship of VGICs can be used to derive the net free energy change (ΔGnet) associated with their voltage-dependent activation. By combining this approach with mutant cycle analysis, it is possible to identify residues that are energetically coupled and contribute to the activation process. Here, we have undertaken a systematic analysis of contact pairs at the interface between the voltage-sensor and pore domains of Shaker Potassium Channel in order to gain insight into how an initial signal can propagate from one region of the Channel to another and trigger the opening of the Channel gate. Our results will be discussed in the context of overall molecular mechanism of electromechanical coupling in voltage-gated ion Channels.
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Congruent Pattern of Accessibility within the Pore of a Voltage-Gated Na+ Channel
Biophysical Journal, 2014Co-Authors: Kevin Oelstrom, ChandaAbstract:Voltage-gated ion Channels open a gate in response to membrane depolarization which allows ions to pass through the transmembrane pore of the Channel. MTSET accessibility studies within the Shaker Potassium Channel revealed that K+ permeation is controlled by an intracellular gate, whereas Cd2+ and Ag+ accessibility measurements in the cyclic nucleotide-gated Channel suggests that this process occurs at the selectivity filter. Modification data of cysteine residues introduced into the DIV S6 of a fast-inactivation removed voltage-gated sodium Channel (VGSC) by MTSET indicate that sodium Channel gating is also regulated by an intracellular gate. However, unlike the Shaker Potassium Channel, VGSCs are not composed of four identical subunits. Despite sequence similarity between each S6 helix, the multi-domain nature of VGSCs implies that there may be asymmetry within the pore that may have consequences in regard to Channel gating. Thus, we sought to determine if analogous positions within the S6 helices of a VGSC act together to form an intracellular gate which occludes the pore while Channels are closed. We scanned the MTSET accessibility of substituted cysteines in the pore lining helices of the first three domains (DI-DIII) in the rat skeletal muscle sodium Channel. The modification data at these sites, selected on the basis of sequence alignment and MTSET accessibility data of DIV-S6, confirms that DII and DIII follow the same pattern of accessibility as that of DIV; however, the extent of block is quite varied across each domain. Along with DI accessibility data, these findings will be discussed in the context of pore gating in related voltage-gated ion Channels.
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Generalized Interaction Energy Analysis of Intersubunit Linkage in Shaker Potassium Channels
Biophysical Journal, 2014Co-Authors: Sandipan Chowdhury, Benjamin M. Haehnel, ChandaAbstract:Voltage-dependent Potassium Channels are crucial for electrical excitability and cellular signaling; however, the molecular machinery that the Channel employs, to relay the state of the voltage sensor to the pore, is not well understood. To gain insight into this voltage-transduction pathway, interacting networks need to be reliably mapped. Here we present a methodology to estimate the strength of site specific interactions called the Generalized Interaction-energy Analysis (or GIA). Our approach involves combining thermodynamic cycle analysis with information from the gating charge verses voltage curves of putative interactors. This methodology was benchmarked against well established kinetic models of Shaker Potassium Channels and BK Channels using Monte Carlo like sampling. Our simulations show that GIA can provide free energy estimates in a self-consistent manner that will be useful to identify site-specific interactors that contribute to gating transitions. Implementing this approach on the Shaker Potassium Channel, we identify a cluster of highly conserved residues, located in the intracellular side of the Channel pore, by the gate, that are energetically coupled. Specifically, it appears that tyrosine 485, on the S6 helix, is critical for maintaining the flexibility of an important hinge in the electromechanical coupling pathway.
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Mapping the Structural Dynamics Within the Shaker Potassium Channel using Tethered Spectroscopic Probes as Chemical Calipers
Biophysical Journal, 2012Co-Authors: Brian W. Jarecki, Suqing Zheng, Xin Zhou, Alessandro Senes, Weiping Tang, ChandaAbstract:Fluorescence based techniques have the distinct advantage of being able to optically probe dynamic structural changes on a biologically relevant timescale. Here we describe the development and application of a novel approach to estimate molecular distances between two sites in a protein. Distance information was inferred by measuring accessibility of a fluorescent tag on one site to a variable length collisional quencher covalently affixed to a second site. The functionalized variable length quenchers therefore act as “tape measures” which can be utilized to obtain point-to-point distance information in the protein structure. Our approach was calibrated using model polyproline peptide substrates of variying lengths. We next tested the biological utility of our approach in a protein of known structure, the Shaker Potassium Channel. Extracellular sites [S1-S2 (T276); S3-S4 (M356); S5-S6 (E422)] within mobile components of the Channel were fluorescently labeled. The distance of the fluorophore at these positions relative to the central pore was estimated by using a series of compounds containing a nitroxide radical separated from a tetraethylammonium moiety (TEA) by variable length PEG spacers. Armed with this Potassium Channel-specific tape measure, cut-open voltage-clamp fluorimetry was utilized to obtain molecular distance estimates of the closed and open Channel. We observed that fluorescence-quenching of labeled residues was tightly correlated with the length of the PEG spacer and the respective distance between the TEA binding site and fluorophore attachment site. The distances estimated from our measurements provide constraints for generating structural models of the closed Channel and may prove useful in mapping sub-nanometer distance changes in the protein structure.
Stefan H. Heinemann - One of the best experts on this subject based on the ideXlab platform.
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Macroscopic Na+ currents in the "Nonconducting" Shaker Potassium Channel mutant W434F.
The Journal of general physiology, 1998Co-Authors: John G. Starkus, Lioba Kuschel, Martin D. Rayner, Stefan H. HeinemannAbstract:C-type inactivation in Shaker Potassium Channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker Channel, such that C-type inactivated Channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F Channels. These conclusions predict that permanently C-type inactivated W434F Channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker Channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated Channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F Channels can be markedly altered by changes in ionic conditions.
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macroscopic na currents in the nonconducting Shaker Potassium Channel mutant w434f
The Journal of General Physiology, 1998Co-Authors: John G. Starkus, Lioba Kuschel, Martin D. Rayner, Stefan H. HeinemannAbstract:C-type inactivation in Shaker Potassium Channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker Channel, such that C-type inactivated Channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F Channels. These conclusions predict that permanently C-type inactivated W434F Channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker Channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated Channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F Channels can be markedly altered by changes in ionic conditions.