Major transmembrane movement associated with colicin Ia channel gating

Colicin Ia, a bacterial protein toxin of 626 amino acid residues, forms voltage-dependent channels in planar lipid bilayer membranes. We have exploited the high affinity binding of streptavidin to biotin to map the topology of the channel-forming domain (roughly 175 residues of the COOH-terminal end) with respect to the membrane. That is, we have determined, for the channel's open and closed states, which parts of this domain are exposed to the aqueous solutions on either side of the membrane and which are inserted into the bilayer. This was done by biotinylating cysteine residues introduced by site-directed mutagenesis, and monitoring by electrophysiological methods the effect of streptavidin addition on channel behavior. We have identified a region of at least 68 residues that flips back and forth across the membrane in association with channel opening and closing. This identification was based on our observations that for mutants biotinylated in this region, streptavidin added to the cis (colicin- containing) compartment interfered with channel opening, and trans streptavidin interfered with channel closing. (If biotin was linked to the colicin by a disulfide bond, the effects of streptavidin on channel closing could be reversed by detaching the streptavidin-biotin complex from the colicin, using a water-soluble reducing agent. This showed that the cysteine sulfur, not just the biotin, is exposed to the trans solution). The upstream and downstream segments flanking the translocated region move into and out of the bilayer during channel opening and closing, forming two transmembrane segments. Surprisingly, if any of several residues near the upstream end of the translocated region is held on the cis side by streptavidin, the colicin still forms voltage-dependent channels, indicating that a part of the protein that normally is fully translocated across the membrane can become the upstream transmembrane segment. Evidently, the identity of the upstream transmembrane segment is not crucial to channel formation, and several open channel structures can exist.     

Topography of diphtheria Toxin's T domain in the open channel state

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a "double dagger" model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed "daggers," TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of approximately 70 residues, is translocated across the membrane to the trans side.     

Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel

TEA is a classical blocker of K(+) channels. From mutagenesis studies, it has been shown that external blockade by TEA is strongly dependent upon the presence of aromatic residue at Shaker position 449 which is located near the extracellular entrance to the pore (Heginbotham, L., and R. MacKinnon. 1992. Neuron. 8:483-491). The data suggest that TEA interacts simultaneously with the aromatic residues of the four monomers. The determination of the 3-D structure of the KcsA channel using X-ray crystallography (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) has raised some issues that remain currently unresolved concerning the interpretation of these observations. In particular, the center of the Tyr82 side chains in KcsA (corresponding to position 449 in Shaker) forms a square of 11.8-A side, a distance which is too large to allow simultaneous interactions of a TEA molecule with the four aromatic side chains. In this paper, the external blockade by TEA is explored by molecular dynamics simulations of an atomic model of KcsA in an explicit phospholipid bilayer with aqueous salt solution. It is observed, in qualitative accord with the experimental results, that TEA is stable when bound to the external side of the wild-type KcsA channel (with Tyr82), but is unstable when bound to a mutant channel in which the tyrosine residue has been substituted by a threonine. The free energy profile of TEA relative to the pore is calculated using umbrella sampling simulations to characterize quantitatively the extracellular blockade. It is found, in remarkable agreement with the experiment, that the TEA is more stably bound by 2.3 kcal/mol to the channel with four tyrosine residues. In the case of the wild-type KcsA channel, TEA (which has the shape of a flattened oblate spheroid) acts as an ideal plug blocking the pore. In contrast, it is considerably more off-centered and tilted in the case of the mutant channel. The enhanced stability conferred by the tyrosine residues does not arise from Pi-cation interactions, but appears to be due to differences in the hydration structure of the TEA. Finally, it is shown that the experimentally observed voltage dependence of TEA block, which is traditionally interpreted in terms of the physical position of the TEA along the axis of the pore, must arise indirectly via coupling with the ions in the pore.     

Analyzing topography of membrane-inserted diphtheria toxin T domain using BODIPY-streptavidin: at low pH, helices 8 and 9 form a transmembrane hairpin but helices 5-7 form stable nonclassical inserted segments on the cis side of the bilayer

Low pH-induced membrane insertion by diphtheria toxin T domain is crucial for A chain translocation into the cytoplasm. To define the membrane topography of the T domain, the exposure of biotinylated Cys residues to the cis and trans bilayer surfaces was examined using model membrane vesicles containing a deeply inserted T domain. To do this, the reactivity of biotin with external and vesicle-entrapped BODIPY-labeled streptavidin was measured. The T domain was found to insert with roughly 70-80% of the molecules in the physiologically relevant orientation. In this orientation, residue 349, located in the loop between hydrophobic helices 8 and 9, was exposed to the trans side of the bilayer, while other solution-exposed residues along the hydrophobic helices 5-9 region of the T domain located near the cis surface. A protocol developed to detect the movement of residues back and forth across the membranes demonstrated that T domain sequences did not rapidly equilibrate between the cis and the trans sides of the bilayer. Binding streptavidin to biotinylated residues prior to membrane insertion only inhibited T domain pore formation for residues in the loop between helices 8 and 9. Pore formation experiments used an approach avoiding interference from transient membrane defects/leakage that may occur upon the initial insertion of protein. Combined, these results indicate that at low pH hydrophobic helices 8 and 9 form a transmembrane hairpin, while hydrophobic helices 5-7 form a nonclassical deeply inserted nontransmembraneous state. We propose that this represents a novel pre-translocation state that is distinct from a previously defined post-translocation state.     

KcsA crystal structure as framework for a molecular model of the Na(+) channel pore

The crystal structure of the pore-forming part of the KcsA bacterial K(+)-selective channel suggests a possible motif for related voltage-gated channels. We examined the hypothesis that the spacial orientation of the KcsA M1 and M2 alpha-helices also predicts the backbone location of S5 and S6 helices of the voltage-gated Na(+) channel. That channel's P region structure is expected to be different because selectivity is determined by side-chain interactions rather than by main-chain carbonyls, and its outer vestibule accommodates relatively large toxin molecules, tetrodotoxin (TTX) and saxitoxin (STX), which interact with selectivity ring residues. The Na(+) channel P loop was well-modeled by the alpha-helix-turn-beta-strand motif, which preserves the relationships for toxin interaction with the Na(+) channel found experimentally. This outer vestibule was docked into the extracellular part of the inverted teepee structure formed by the S5 and S6 helices that were spacially located by coordinates of the KcsA M1 and M2 helix main chains [Doyle et al. (1998) Science 280, 69-74], but populated with side chains of the respective S5 and S6 structures. van der Waals contacts were optimized with minimal adjustment of the S5, S6, and P loop structures, forming a densely packed pore structure. Nonregular external S5-P and P-S6 segments were not modeled here, except the P-S6 segment of domain II. The resulting selectivity region structure is consistent with Na(+) channel permeation properties, offering suggestions for the molecular processes involved in selectivity. The ability to construct a Na(+) channel pore model consistent with most of the available biophysical and mutational information suggests that the KcsA structural framework may be conserved in voltage-gated channels.     

Identification of a translocated gating charge in a voltage-dependent channel. Colicin E1 channels in planar phospholipid bilayer membranes.

The availability of primary sequences for ion-conducting channels permits the development of testable models for mechanisms of voltage gating. Previous work on planar phospholipid bilayers and lipid vesicles indicates that voltage gating of colicin E1 channels involves translocation of peptide segments of the molecule into and across the membrane. Here we identify histidine residue 440 as a gating charge associated with this translocation. Using site-directed mutagenesis to convert the positively charged His440 to a neutral cysteine, we find that the voltage dependence for turn-off of channels formed by this mutant at position 440 is less steep than that for wild-type channels; the magnitude of the change in voltage dependence is consistent with residue 440 moving from the trans to the cis side of the membrane in association with channel closure. The effect of trans pH changes on the ion selectivity of channels formed by the carboxymethylated derivative of the cysteine 440 mutant independently establishes that in the open channel state, residue 440 lies on the trans side of the membrane. On the basis of these results, we propose that the voltage-gated opening of colicin E1 channels is accompanied by the insertion into the bilayer of a helical hairpin loop extending from residue 420 to residue 459, and that voltage-gated closing is associated with the extrusion of this loop from the interior of the bilayer back to the cis side.     

Antibody interaction with the amphotericin channel

The monoclonal antibodies against asymmetric channel formed in the lipid bilayer of polyene antibiotic amphotericin B and cholesterol after addition of the antibiotic to the compartment from the cis side of the membrane were obtained. The effect of the antibodies on ion conductance of the channel depends on the distribution of cholesterol in the membrane. When cholesterol was present on both sides of the lipid bilayer, three antibody molecules bound to the channel from the trans side of the membrane, thus markedly increasing the lifetime of the open state of the channel. When cholesterol was present in the cis monolayer only, the antibodies, added to the trans compartment of the cell, reduced the membrane conduction.     

Binding of correolide to the K(v)1.3 potassium channel: characterization of the binding domain by site-directed mutagenesis

Correolide is a novel immunosuppressant that inhibits the voltage-gated potassium channel K(v)1.3 [Felix et al. (1999) Biochemistry 38, 4922-4930]. [(3)H]Dihydrocorreolide (diTC) binds with high affinity to membranes expressing homotetrameric K(v)1.3 channels, and high affinity diTC binding can be conferred to the diTC-insensitive channel, K(v)3.2, after substitution of three nonconserved residues in S(5) and S(6) with the corresponding amino acids present in K(v)1.3 [Hanner et al. (1999) J. Biol. Chem. 274, 25237-25244]. Site-directed mutagenesis along S(5) and S(6) of K(v)1.3 was employed to identify those residues that contribute to high affinity binding of diTC. Binding of monoiodotyrosine-HgTX(1)A19Y/Y37F ([(125)I]HgTX(1)A19Y/Y37F) in the external vestibule of the channel was used to characterize each mutant for both tetrameric channel formation and levels of channel expression. Substitutions at Leu(346) and Leu(353) in S(5), and Ala(413), Val(417), Ala(421), Pro(423), and Val(424) in S(6), cause the most dramatic effect on diTC binding to K(v)1.3. Some of the critical residues in S(6) appear to be present in a region of the protein that alters its conformation during channel gating. Molecular modeling of the S(5)-S(6) region of K(v)1.3 using the X-ray coordinates of the KcsA channel, and other experimental constraints, yield a template that can be used to dock diTC in the channel. DiTC appears to bind in the water-filled cavity below the selectivity filter to a hydrophobic pocket contributed by the side chains of specific residues. High affinity binding is predicted to be determined by the complementary shape between the bowl-shape of the cavity and the shape of the ligand. The conformational change that occurs in this region of the protein during channel gating may explain the state-dependent interaction of diTC with K(v)1.3.     

Heparin influence on alpha-staphylotoxin formed channel

The effects of heparin on ion channels formed by Staphylococcus aureus alpha-toxin (ST channel) in lipid bilayers were studied under voltage clamp conditions. Heparin concentrations as small as 100 pM induced a sharp dose-dependent increase in channel voltage sensitivity. This was only observed when heparin was added to the negative-potential side of lipid bilayers in the presence of divalent cations. Divalent cations differ in their efficiency: Zn2+>Ca2+>Mg2+. The apparent positive gating charge increased 2-3-fold with heparin addition as well as with acidification of the bathing solution. 'Free' carboxyl groups and carboxyl groups in ion pairs of the protein moiety are hypothesized to interact with sulfated groups of heparin through divalent cation bridges. The cis mouth of the channel (that protrudes beyond the membrane plane on the side of ST addition and to which voltage was applied) is less sensitive to heparin than the trans-mouth. It is suggested that charged residues which interact with heparin at the cis mouth of ST channels and which contribute to the effective gating charge at negative voltage may be physically different from those at the trans mouth and at positive voltage.     

Mechanism of Cd(2+) Coordination during Slow Inactivation in Potassium Channels

In K(+) channels, rearrangements of the pore outer vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggests these movements to be modest in magnitude. In this study, we show that under physiological conditions, the KcsA outer vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd(2+) enhances the rate of C-type inactivation in an cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K(+) channels demonstrate that these Cd(2+) metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd(2+)-bound Y82C-KcsA in the closed state, together with electron paramagnetic resonance distance measurements in the KcsA outer vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation.     

Sequence-function analysis of the K+-selective family of ion channels using a comprehensive alignment and the KcsA channel structure

Sequence-function analysis of K(+)-selective channels was carried out in the context of the 3.2 A crystal structure of a K(+) channel (KcsA) from Streptomyces lividans (Doyle et al., 1998). The first step was the construction of an alignment of a comprehensive set of K(+)-selective channel sequences forming the putative permeation path. This pathway consists of two transmembrane segments plus an extracellular linker. Included in the alignment are channels from the eight major classes of K(+)-selective channels from a wide variety of species, displaying varied rectification, gating, and activation properties. Segments of the alignment were assigned to structural motifs based on the KcsA structure. The alignment's accuracy was verified by two observations on these motifs: 1), the most variability is shown in the turret region, which functionally is strongly implicated in susceptibility to toxin binding; and 2), the selectivity filter and pore helix are the most highly conserved regions. This alignment combined with the KcsA structure was used to assess whether clusters of contiguous residues linked by hydrophobic or electrostatic interactions in KcsA are conserved in the K(+)-selective channel family. Analysis of sequence conservation patterns in the alignment suggests that a cluster of conserved residues is critical for determining the degree of K(+) selectivity. The alignment also supports the near-universality of the "glycine hinge" mechanism at the center of the inner helix for opening K channels. This mechanism has been suggested by the recent crystallization of a K channel in the open state. Further, the alignment reveals a second highly conserved glycine near the extracellular end of the inner helix, which may be important in minimizing deformation of the extracellular vestibule as the channel opens. These and other sequence-function relationships found in this analysis suggest that much of the permeation path architecture in KcsA is present in most K(+)-selective channels. Because of this finding, the alignment provides a robust starting point for homology modeling of the permeation paths of other K(+)-selective channel classes and elucidation of sequence-function relationships therein. To assay these applications, a homology model of the Shaker A channel permeation path was constructed using the alignment and KcsA as the template, and its structure evaluated in light of established structural criteria.     

Mapping the membrane topography of the TH6–TH7 segment of the diphtheria toxin T-domain channel

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1–TH4, translocates across the membrane to the trans side. Residues near either end of the TH6–TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6–TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6–TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue’s accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6–TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.     

Structural basis of the KcsA K(+) channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method

We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K(+) channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K(+) channel. Based on properties of the AgTx2 binding site with the KcsA K(+) channel, we present a surface motif that is observed in pore-blocking toxins affecting the K(+) channel. Furthermore, we also explain the structural basis of the specificity of the K(+) channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.     

Molecular dynamics of the KcsA K(+) channel in a bilayer membrane

Molecular dynamics (MD) simulations of an atomic model of the KcsA K(+) channel embedded in an explicit dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer solvated by a 150 mM KCl aqueous salt solution are performed and analyzed. The model includes the KcsA K(+) channel, based on the recent crystallographic structure of, Science. 280:69-77), 112 DPPC, K(+) and Cl(-) ions, as well as over 6500 water molecules for a total of more than 40,000 atoms. Three K(+) ions are explicitly included in the pore. Two are positioned in the selectivity filter on the extracellular side and one in the large water-filled cavity. Different starting configurations of the ions and water molecules in the selectivity filter are considered, and MD trajectories are generated for more than 4 ns. The conformation of KcsA is very stable in all of the trajectories, with a global backbone root mean square (RMS) deviation of less than 1.9 A with respect to the crystallographic structure. The RMS atomic fluctuations of the residues surrounding the selectivity filter on the extracellular side of the channel are significantly lower than those on the intracellular side. The motion of the residues with aromatic side chains surrounding the selectivity filter (Trp(67), Trp(68), Tyr(78), and Tyr(82)) is anisotropic with the smallest RMS fluctuations in the direction parallel to the membrane plane. A concerted dynamic transition of the three K(+) ions in the pore is observed, during which the K(+) ion located initially in the cavity moves into the narrow part of the selectivity filter, while the other two K(+) ions move toward the extracellular side. A single water molecule is stabilized between each pair of ions during the transition, suggesting that each K(+) cation translocating through the narrow pore is accompanied by exactly one water molecule, in accord with streaming potential measurements (, Biophys. J. 55:367-371). The displacement of the ions is coupled with the structural fluctuations of Val(76) and Gly(77), in the selectivity filter, as well as the side chains of Glu(71), Asp(80), and Arg(89), near the extracellular side. Thus the mechanical response of the channel structure at distances as large as 10-20 A from the ions in the selectivity filter appears to play an important role in the concerted transition.     

A protein-conducting channel in the endoplasmic reticulum

The existence of a protein-conducting channel in the endoplasmic reticulum membrane was demonstrated by electrophysiological techniques. Pancreatic rough microsome (RM) vesicles were fused to one side (cis) of a planar lipid bilayer separating two aqueous compartments of 50 mM salt. This exposed the cytoplasmic surface of the RMs, with its attached ribosomes, to the cis chamber. Addition of 100 microM puromycin to the cis side caused a large increase in membrane conductance, presumably the result of puromycin-induced clearance of nascent protein chains from the lumen of protein-conducting channels. When puromycin was added at low concentrations (0.33 microM), single channels of 220 pS were observed. These closed when the salt concentration was raised to levels at which ribosomes detach from the membrane (150-400 mM), indicating that the attached ribosome keeps the channel in an open conformation. A mechanism for a complete cycle of opening and closing of the protein-conducting channel is suggested.     

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl⁺) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H⁺). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C_18:1 to C_22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H⁺ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H⁺ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA

Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.     

Contribution of ion binding affinity to ion selectivity and permeation in KcsA, a model potassium channel

Ion permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K(+) to this mutant reveals a single set of low-affinity K(+) binding sites, similar to that seen in the binding of K(+) to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K(+) permeation in E71A. Nonetheless, the E71A mutant retains K(+) selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K(+) and also conducts Na(+). Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.     

Interactions of phospholipids with the potassium channel KcsA

The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.