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Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion-bound configuration (i.e., water, K + , water, K + -ion-bound configuration) of the K + channel's selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type-like ion selectivity and apparent K + -binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K + channel's selectivity filter, which fully agrees with the water-K + -ion-coupled transport detected by streaming potential measurements., Competing Interests: The authors declare no conflict of interest.

The Erwinia chrysanthemi ligand-gated ion channel, ELIC, is considered an excellent structural and functional surrogate for the whole pentameric ligand-gated ion channel family. Despite its simplicity, ELIC is structurally capable of undergoing ligand-dependent activation and a concomitant desensitization process. To determine at the molecular level the structural changes underlying ELIC's function, it is desirable to produce large quantities of protein. This protein should be properly folded, fully-functional and amenable to structural determinations. In the current paper, we report a completely new protocol for the expression and purification of milligram quantities of fully-functional, more stable and crystallizable ELIC. The use of an autoinduction media and inexpensive detergents during ELIC extraction, in addition to the high-quality and large quantity of the purified channel, are the highlights of this improved biochemical protocol., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mode-shift or hysteresis has been reported in ion channels. Voltage-shift for gating currents is well documented for voltage-gated cation channels (VGCC), and it is considered a voltage-sensing domain's (VSD) intrinsic property. However, uncoupling the Shaker K + channel's pore domain (PD) from the VSD prevented the mode-shift of the gating currents. Consequently, it was proposed that an open-state stabilization of the PD imposes a mechanical load on the VSD, which causes its mode-shift. Furthermore, the mode-shift displayed by hyperpolarization-gated cation channels is likely caused by structural changes at the channel's PD similar to those underlying C-type inactivation. To demonstrate that the PD of VGCC undergoes hysteresis, it is imperative to study its gating process in the absence of the VSD. A back-door strategy is to use KcsA (a K + channel from the bacteria Streptomyces lividans ) as a surrogate because it lacks a VSD and exhibits an activation coupled to C-type inactivation. By directly measuring KcsA's activation gate opening and closing in conditions that promote or halt C-type inactivation, we have found ( i ) that KcsA undergoes mode-shift of gating when having K + as the permeant ion; ( ii ) that Cs + or Rb + , known to halt C-inactivation, prevented mode-shift of gating; and ( iii ) that, in the total absence of C-type inactivation, KcsA's mode-shift was prevented. Finally, our results demonstrate that an allosteric communication causes KcsA's activation gate to "remember" the conformation of the selectivity filter, and hence KcsA requires a different amount of energy for opening than for closing.

KcsA, the bacterial K(+) channel from Streptomyces lividans, is the prototypical model system to study the functional and structural correlations of the pore domain of eukaryotic voltage-gated K(+) channels (Kv channels). It contains all the molecular elements responsible for ion conduction, activation, deactivation and inactivation gating [1]. KcsA's structural simplicity makes it highly amenable for structural studies. Therefore, it is methodological advantageous to produce large amounts of functional and properly folded KcsA in a cost-effective manner. In the present study, we show an optimized protocol for the over-expression and purification of large amounts of high-quality, fully functional and crystallizable KcsA using inexpensive detergents, which significantly lowered the cost of the purification process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Using a constitutively active channel mutant, we solved the structure of full-length KcsA in the open conformation at 3.9 Å. The structure reveals that the activation gate expands about 20 Å, exerting a strain on the bulge helices in the C-terminal domain and generating side windows large enough to accommodate hydrated K(+) ions. Functional and spectroscopic analysis of the gating transition provides direct insight into the allosteric coupling between the activation gate and the selectivity filter. We show that the movement of the inner gate helix is transmitted to the C-terminus as a straightforward expansion, leading to an upward movement and the insertion of the top third of the bulge helix into the membrane. We suggest that by limiting the extent to which the inner gate can open, the cytoplasmic domain also modulates the level of inactivation occurring at the selectivity filter.

Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K(+) channel KcsA shows three distinct gating modes, high-P(o), low-P(o) and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. Here we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side chain-specific way. These gating modes mirror those seen in wild-type channels and suggest that specific interactions in the side chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. The present results highlight the key role of the selectivity filter in regulating modal gating behavior in K(+) channels.

The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

Interconversion between conductive and non-conductive forms of the K(+) channel selectivity filter underlies a variety of gating events, from flicker transitions (at the microsecond timescale) to C-type inactivation (millisecond to second timescale). Here we report the crystal structure of the Streptomyces lividans K(+) channel KcsA in its open-inactivated conformation and investigate the mechanism of C-type inactivation gating at the selectivity filter from channels 'trapped' in a series of partially open conformations. Five conformer classes were identified with openings ranging from 12 A in closed KcsA (Calpha-Calpha distances at Thr 112) to 32 A when fully open. They revealed a remarkable correlation between the degree of gate opening and the conformation and ion occupancy of the selectivity filter. We show that a gradual filter backbone reorientation leads first to a loss of the S2 ion binding site and a subsequent loss of the S3 binding site, presumably abrogating ion conduction. These structures indicate a molecular basis for C-type inactivation in K(+) channels.

The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117-122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al. identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

K+ channels undergo a time-dependent slow inactivation process that plays a key role in modulating cellular excitability. Here we show that in the prokaryotic proton-gated K+ channel KcsA, the number and strength of hydrogen bonds between residues in the selectivity filter and its adjacent pore helix determine the rate and extent of C-type inactivation. Upon channel activation, the interaction between residues at positions Glu71 and Asp80 promotes filter constriction parallel to the permeation pathway, which affects K+-binding sites and presumably abrogates ion conduction. Coupling between these two positions results in a quantitative correlation between their interaction strength and the stability of the inactivated state. Engineering of these interactions in the eukaryotic voltage-dependent K+ channel Kv1.2 suggests that a similar mechanistic principle applies to other K+ channels. These observations provide a plausible physical framework for understanding C-type inactivation in K+ channels.

We show that in the potassium channel KcsA, proton-dependent activation is followed by an inactivation process similar to C-type inactivation, and this process is suppressed by an E71A mutation in the pore helix. EPR spectroscopy demonstrates that the inner gate opens maximally at low pH regardless of the magnitude of the single-channel-open probability, implying that stationary gating originates mostly from rearrangements at the selectivity filter. Two E71A crystal structures obtained at 2.5 A reveal large structural excursions of the selectivity filter during ion conduction and provide a glimpse of the range of conformations available to this region of the channel during gating. These data establish a mechanistic basis for the role of the selectivity filter during channel activation and inactivation.

The molecular architecture of the NH(2) and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 5-24 and 121-160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (DeltaHo(-1)), accessibility parameters (PiO(2) and PiNiEdda), and inter-subunit spin-spin interaction (Omega) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH(2) terminus of KcsA forms an alpha-helix anchored at the membrane-water interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 40-50 A towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.

The intramembrane molecular events underlying activation gating in the Streptomyces K+ channel were investigated by site-directed spin-labeling methods and electron paramagnetic resonance spectroscopy. A comparison of the closed and open conformations of the channel revealed periodic changes in spin-label mobility and intersubunit spin-spin interaction consistent with rigid-body movements of the two transmembrane helices TM1 and TM2. These changes involve translations and counterclockwise rotations of both helices relative to the center of symmetry of the channel. The movement of TM2 increases the diameter of the permeation pathway along the point of convergence of the four subunits, thus opening the pore. Although the extracellular residues flanking the selectivity filter remained immobile during gating, small movements were detected at the C-terminal end of the pore helix, with possible implications to the gating mechanism.

The transmembrane organization of a potassium channel from Streptomyces lividans has been studied using site-directed spin labeling techniques and electron paramagnetic resonance spectroscopy. In the tetrameric channel complex, two alpha-helices were identified per monomer and assigned to the amino acid sequence. Probe mobility and accessibility data clearly establish that the first helix (TM1) is located in the perimeter of the channel, showing extensive protein-lipid contacts, while the second helix (TM2) is closer to the four-fold symmetric axis of the channel, lining the intracellular vestibule. A large conformational change in the C-terminal end of TM2 was measured when comparing conditions that favor either the open or closed states. The present data suggest that the diameter of the internal vestibule increases with channel opening.

Because of its size, high levels of expression, and unusual detergent stability, the small K+ channel from Streptomyces lividans (SKC1) is considered to be an ideal candidate for detailed structural analysis. In this paper, we have used planar lipid bilayers and radiotracer uptake experiments to study purified and reconstituted SKC1, in an attempt to develop a bulk assay for its functional characterization. In channels reconstituted into liposomes with external pH 3.5 and intravesicular pH 7.5, a time-dependent SKC1-catalyzed 86Rb+ uptake was observed. This cationic influx was blocked by Ba2+ ions with a Ki (external) of 0.4 mM and was shown to have the following selectivity sequence: K+ > Rb+ > NH4+ >> Na+ > Li+. In experiments with external pH 7.5 or in liposomes containing no channels, no 86Rb+ uptake was detected. When SKC1 was incorporated into planar lipid bilayers, we failed to observe significant single-channel activity at neutral pH but detected frequent multiple-channel openings a pH < 5.0. These results indicate that under these experimental conditions SKC1 behaves as a pH-gated K+ channel in which protonation of one or more residues promotes channel opening. At acidic pH and symmetrical 200 mM KCl solutions, SKC1 showed numerous brief openings with a main single-channel conductance of 135 pS and a subconductance state of 70 pS. Channel open probability showed a slight voltage dependence, with higher activities observed at negative potentials, a fact which may suggest that the protonation site lies within the transmembrane electrical field. Attempts to determine the pKa of channel activation were obscured by intrinsic limitations of the 86Rb+ flux assay. However, it appears to be lower than pH 4.0. Limited proteolysis experiments demonstrated that SKC1 reconstitutes vectorially, almost exclusively in the right-side-out configuration, indicating that the protonation site responsible for channel opening is located at the extracellular face of the channel. These results point toward a potentially novel gating mechanism for SKC1 and open the possibility of using transmembrane-driven radiotracer influx experiments as a reliable bulk functional assay for reconstituted SKC1.

Fourier transform infrared (FTIR) spectroscopy was used to probe the secondary structure, orientation, and the kinetics of amide hydrogen-deuterium exchange (HX) of the small K+ channel from Streptomyces lividans. Frequency component analysis of the amide I band showed that SKC1 is composed of 44-46% alpha-helix, 21-24% beta-sheet, 10-12% turns and 18-20% unordered structures. The order parameter S of the helical component of SKC1 was between 0.60 and 0.69. Close to 80% of SKC1 amide protons exchange within approximately 3 h of D2O exposure, suggesting that the channel is largely accessible to solvent exchange. These results are consistent with a model of SKC1 in which helices slightly tilted from the membrane normal line the water-filled vestibules that flank the K+ selectivity filter.

SKC1, a 160-residue potassium channel with two putative transmembrane (TM) segments was recently identified from Streptomyces lividans. Its high levels of expression, small size, and ease of purification make SKC1 an ideal candidate for high-resolution structural studies. We have initiated the structural characterization of this channel by assessing its oligomeric behavior, stability in detergent, general hydrodynamic properties, and preliminary secondary structure content. SKC1 was readily expressed and purified to homogeneity by sequential metal-chelate and gel filtration chromatography. Standard SDS-PAGE, together with chemical cross-linking analysis indicated that SKC1 behaves as a tightly associated tetramer even in the presence of SDS. Using a gel shift assay to assess its oligomeric state, we determined that SKC1 is stable as a tetramer in most detergents and can be maintained in nonionic detergent solutions for extended periods of time. The tetramer is also stable at relatively high temperatures, with an oligomer-to-monomer transition occurring at approximately 65 degrees C. The Stokes radius of the micellar complex is 5 nm as determined from gel filtration chromatography of SKC1 in dodecyl maltoside. Preliminary estimations of secondary structure from CD spectroscopy showed that the channel exists mostly in alpha-helical conformation, with more than 50% alpha-helical, close to 20% beta-sheet, 10% beta-turn, and about 15% unassigned or random coil. These results are consistent with the idea that a bundle of alpha-helices forming a tetramer around the ion-conductive pathway is the common structural motif for members of the voltage-dependent channel superfamily.