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TOKs are outwardly rectifying K+ channels in fungi with two pore-loops and eight transmembrane spans. Here, we describe the TOKs from four pathogens that cause the majority of life-threatening fungal infections in humans. These TOKs pass large currents only in the outward direction like the canonical isolate from Saccharomyces cerevisiae (ScTOK), and distinct from other K+ channels. ScTOK, AfTOK1 (Aspergillus fumigatus), and H99TOK (Cryptococcus neoformans grubii) are K+ -selective and pass current above the K+ reversal potential. CaTOK (Candida albicans) and CnTOK (Cryptococcus neoformans neoformans) pass both K+ and Na+ and conduct above a reversal potential reflecting the mixed permeability of their selectivity filter. Mutations in CaTOK and ScTOK at sites homologous to those that open the internal gates in classical K+ channels are shown to produce inward TOK currents. A favored model for outward rectification is proposed whereby the reversal potential determines ion occupancy, and thus, conductivity, of the selectivity filter gate that is coupled to an imperfectly restrictive internal gate, permitting the filter to sample ion concentrations on both sides of the membrane.

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with Ki of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the alpha-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an alpha-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

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 [...]

The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region. We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.

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 [...]

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.

A study is carried out on the regulated gating through the direct binding of ligands by large cytoplasmic RCK (regulates conductance of K(super +)) domains in a subset of K(super +) channels and further investigations are done on the role of RCK domain is done. The findings of the study support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.

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]