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Interconversion between conductive and non-conductive forms of the [K.sup.+] 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.sup.+] 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 Å in closed KcsA (Cα-Cα distances at Thr 112) to 32 Å 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.sup.+] channels., The functional behaviour of most ion channels is defined by the relationship between two coupled mechanisms: activation and inactivation gating. Activation is associated with a large hinged motion around the [...]

The coupled interplay between activation and inactivation gating is a functional hallmark of [K.sup.+] channels (1,2). This coupling has been experimentally demonstrated through ion interaction effects (3,4) and cysteine accessibility (1), and is associated with a well defined boundary of energetically coupled residues (2). The structure of the [K.sup.+] channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating (5). 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 Thr74 and Thr75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp67, Glu 71 and Asp 80 to destabilize the selectivity filter (6,7), 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.sup.+] 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.sup.+] channels., The K-channel pore-domain contains all the molecular elements necessary for ion conduction and for activation and inactivation gating (8,9). Activation is classically associated with conformational rearrangements at the inner helix [...]

We have analyzed the local structure and dynamics of the prokaryotic voltage-dependent [K.sup.+] channel (KvAP) at 0 mitlivolts, using site-directed spin labeling and electron paramagnetic resonance spectroscopy. We show that the S4 segment is located at the protein/lipid interface, with most of its charges protected from the lipid environment. Structurally, S4 is highly dynamic and is separated into two short helices by a flexible linker. Accessibility and dynamics data indicate that the S1 segment is surrounded by other parts of the protein. We propose that s1 is at the contact interface between the voltage-sensing and pore domains. These results establish the general principles of voltage-dependent channel structure in a biological membrane., Voltage-dependent channels are composed of two functionally linked but structurally independent domains (1-4). The pore domain is responsible for ion selectivity and contains the channel gate, whereas a voltage-sensing domain [...]

Author(s): Eduardo Perozo (corresponding author) [1]; D. Marien Cortes [1]; Pornthep Sompornpisut [1, 2]; Anna Kloda [3]; Boris Martinac [3] During severe osmotic challenges, the large-conductance mechanosensitive channel (MscL) of [...]

A purified and reconstituted Streptomyces lividans potassium channel (SKC1) was analyzed using lipid bilayer assays and radiotracer influx measurements. Experiments with SKC1 and lipid bilayers at pH less than 5.0 showed multiple-channel openings. A time-dependent cationic influx activated by SKC1 was observed at external pH 3.5 and intravesicular pH 7.5 in liposomes with channels. These suggest that protonation of residues is responsible for channel opening in SKC1 acting as a pH-gated K+ channel.

The structural dynamics of Streptomyces lividans K+ channel (SKC1) was investigated by using stoichiometry and stability analyses. Results of the research revealed that a bundle of alpha-helices forming a tetramer around the ion-conductive pathway is a usual structural formation for voltage-dependent channel superfamily. Furthermore, the oligomeric complexes had a tendency to dissociate under certain conditions.

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. [ABSTRACT FROM AUTHOR]

The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to [O.sub.2] or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of EcoMscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2. KEY WORDS: mechanosensitive channel * solvent accessibility * dipolar coupling * EPR * transmembrane segments

The molecular architecture of the [NH.sub.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 121-100) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility ([[Delta]Ho.sub.-1]), accessibility parameters ([[Pi]O.sub.2] and [Pi]NiEdda), 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.sub.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 [Angstrom] 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. KEY WORDS: KcsA * cytoplasmic domains * EPR spectroscopy * three-dimensional fold

The selectivity filter and the activation gate in potassium channels are functionally and structurally coupled. An allosteric coupling underlies C-type inactivation coupled to activation gating in this ion-channel family (i.e., opening of the activation gate triggers the collapse of the channel's selectivity filter). We have identified the second Threonine residue within the TTVGYGD signature sequence of K+ channels as a crucial residue for this allosteric communication. A Threonine to Alanine substitution at this position was studied in three representative members of the K+-channel family. Interestingly, all of the mutant channels exhibited lack of C-type inactivation gating and an inversion of their allosteric coupling (i.e., closing of the activation gate collapses the channel's selectivity filter). A state-dependent crystallographic study of KcsA-T75A proves that, on activation, the selectivity filter transitions from a nonconductive and deep C-type inactivated conformation to a conductive one. Finally, we provide a crystallographic demonstration that closed-state inactivation can be achieved by the structural collapse of the channel's selectivity filter. [ABSTRACT FROM AUTHOR]

C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA's mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 A°) and deep C-type inactivated (Y82A at 2.4 A°) conformations. These structural snapshots represent KcsA's transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle. [ABSTRACT FROM AUTHOR]