Luis G. Cuello

Molecular determinants of gating at the potassium-channel selectivity filter.

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 Å 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.

Structural mechanism of C-type inactivation in 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 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+ channels.

Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy

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 α-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.

Structural basis for the coupling between activation and inactivation gates 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.

Detection of the Opening of the Bundle Crossing in KcsA with Fluorescence Lifetime Spectroscopy Reveals the Existence of Two Gates for Ion Conduction

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.

Structural dynamics of an isolated voltage-sensor domain in a lipid bilayer.

A strong interplay between the voltage-sensor domain (VSD) and the pore domain (PD) underlies voltage-gated channel functions. In a few voltage-sensitive proteins, the VSD has been shown to function without a canonical PD, although its structure and oligomeric state remain unknown. Here, using EPR spectroscopy, we show that the isolated VSD of KvAP can remain monomeric in a reconstituted bilayer and retain a transmembrane conformation. We find that water-filled crevices extending deep into the membrane around S3, a scaffold conducive to transport of protons/cations, are intrinsic to the VSD. Differences in solvent accessibility in comparison to the full-length KvAP allowed us to define an interacting footprint of the PD on the VSD. This interaction is centered around S1 and S2 and suggests a rotation of 70 degrees -100 degrees relative to Kv1.2-Kv2.1 chimera. Sequence-conservation patterns in Kv channels, Hv channels, and voltage-sensitive phosphatases reveal several near-universal features suggesting a common molecular architecture for all VSDs.

Mechanism of activation gating in the full-length KcsA K+ channel.

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.

A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library

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 α-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 α-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

Gating of the proton-gated ion channel from Gloeobacter violaceus at pH 4 as revealed by X-ray crystallography

Significance Determination of the structure of ion channels in their physiologically relevant states remains a major challenge. Structural models of the unliganded closed-channel and the fully liganded open-channel conformations of different members of the nicotinic-receptor superfamily have been generated using cryoelectron microscopy or X-ray crystallography. In this paper, we describe the structure of what appears to be the closed-channel conformation in its liganded state. We used X-ray crystallography to solve the structure of two mutants of a proton-gated bacterial ortholog that exhibit a reduced equilibrium constant for the closed-to-open transition; to favor the ligand-bound state, the crystals were grown at pH ∼4.0. Compared with the liganded open-channel conformation, the closed-channel conformation presents a narrower pore, but an indistinguishable extracellular domain. Cryoelectron microscopy and X-ray crystallography have recently been used to generate structural models that likely represent the unliganded closed-channel conformation and the fully liganded open-channel conformation of different members of the nicotinic-receptor superfamily. To characterize the structure of the closed-channel conformation in its liganded state, we identified a number of positions in the loop between transmembrane segments 2 (M2) and 3 (M3) of a proton-gated ortholog from the bacterium Gloeobacter violaceus (GLIC) where mutations to alanine reduce the liganded-gating equilibrium constant, and solved the crystal structures of two such mutants (T25′A and Y27′A) at pH ∼4.0. At the level of backbone atoms, the liganded closed-channel model presented here differs from the liganded open-channel structure of GLIC in the pre-M1 linker, the M3–M4 loop, and much more prominently, in the extracellular half of the pore lining, where the more pronounced tilt of the closed-channel M2 α-helices toward the pore’s long axis narrows the permeation pathway. On the other hand, no differences between the liganded closed-channel and open-channel models could be detected at the level of the extracellular domain, where conformational changes are expected to underlie the low-to-high proton-affinity switch that drives gating of proton-bound channels. Thus, the liganded closed-channel model is nearly indistinguishable from the recently described “locally closed” structure. However, because cross-linking strategies (which could have stabilized unstable conformations) and mutations involving ionizable side chains (which could have affected proton-gated channel activation) were purposely avoided, we favor the notion that this structure represents one of the end states of liganded gating rather than an unstable intermediate.

A molecular mechanism for proton-dependent gating in KcsA

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al.[1] 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.