Selectivity Filter Of Potassium Channels Research Articles

Structural Determinants for Rubidium Binding Affinity in the Selectivity Filter of Potassium Channels

Two competing models for potassium permeation has been proposed based on high-resolution crystallographic data, electrophysiological studies of highly K+-selective channels or mostly on computational studies. The two models are named according the way the ions interact within the channel's selectivity filter (SF). The soft knock-on model proposes the existence of two iso-energetic ion bound configurations, known as the 1-3 and 2-4 ion bound configurations. In this model, a water molecule and a K+-ion cross the cell membrane per cycle of transport and the alternate existence of the two ion bound configurations, underlies the ion selective high transport rate of potassium channels.

Potassium channel selectivity filter dynamics revealed by single-molecule FRET.

Potassium (K) channels exhibit exquisite selectivity for conduction of K+ ions over other cations, particularly Na+. High resolution structures reveal an archetypal selectivity filter (SF) conformation in which dehydrated K+, but not Na+, ions are perfectly coordinated. Using single molecule FRET (smFRET), we show that the SF-forming loop (SF-loop) in KirBac1.1 transitions between constrained and dilated conformations as a function of ion concentrations. The constrained conformation, essential for selective K+ permeability, is stabilized by K+ but not Na+ ions. Mutations that render channels non-selective result in dilated and dynamically unstable conformations, independent of the permeant ion. Further, while wild type KirBac1.1 channels are K+-selective in physiological conditions, Na+ permeates in the absence of K+. Moreover, while K+ gradients preferentially support 86Rb+ fluxes, Na+ gradients preferentially support 22Na+ fluxes. This suggests differential ion selectivity in constrained versus dilated states, potentially providing a structural basis for this anomalous mole fraction effect.

Chemical substitutions in the selectivity filter of potassium channels do not rule out constricted-like conformations for C-type inactivation

In many K+ channels, prolonged activating stimuli lead to a time-dependent reduction in ion conduction, a phenomenon known as C-type inactivation. X-ray structures of the KcsA channel suggest that this inactivated state corresponds to a "constricted" conformation of the selectivity filter. However, the functional significance of the constricted conformation has become a matter of debate. Functional and structural studies based on chemically modified semisynthetic KcsA channels along the selectivity filter led to the conclusion that the constricted conformation does not correspond to the C-type inactivated state. The main results supporting this view include the observation that C-type inactivation is not suppressed by a substitution of D-alanine at Gly77, even though this modification is believed to lock the selectivity filter into its conductive conformation, whereas it is suppressed following amide-to-ester backbone substitutions at Gly77 and Tyr78, even though these structure-conserving modifications are not believed to prevent the selectivity filter from adopting the constricted conformation. However, several untested assumptions about the structural and functional impact of these chemical modifications underlie these arguments. To make progress, molecular dynamics simulations based on atomic models of the KcsA channel were performed. The computational results support the notion that the constricted conformation of the selectivity filter corresponds to the functional C-type inactivated state of the KcsA. Importantly, MD simulations reveal that the semisynthetic KcsAD-ala77 channel can adopt an asymmetrical constricted-like nonconductive conformation and that the amide-to-ester backbone substitutions at Gly77 and Tyr78 perturb the hydrogen bonding involving the buried water molecules stabilizing the constricted conformation.

Regulation of ion Permeation in the Selectivity Filter of Potassium Channels

Ion efflux through potassium channels is regulated by a main gate controlled by ligands and/or the transmembrane voltage. The opening of the main gate let K ions flow down their gradient, diffusing through the selectivity filter of the channel, which can also contribute to the regulation of the ionic current by changing its conformation. Functional and structural data have brought evidence of interaction between the main gate and the selectivity filter of potassium channels. It is understood that opening the main gate favors the inactivated state of the filter, whereas closing the main gate stabilize the conducting state of the filter.We have performed molecular dynamics simulations and potential of mean force calculations showing that the selectivity filter of the KcsA channel in its canonical conducting state, as captured by crystallography, does not allow the diffusion of ions at a rate compatible with experimental data. However, our calculations reveal that the opening of the main gate favors a transient conformational state of the selectivity filter that allows transport rate near the ion diffusion limit. The selectivity filter is thus expected to go from a pre-conducting state with high ion binding affinity to a conducting state with lower ion binding affinity, eventually transiting to the inactivated state. The detailed balance of forces involved in the fine regulation of ion permeation is well described by the latest generation of classical force-fields, which can reveal structural features of ion channels with an unprecedented level of precision.

Comprehensive Re-Sequencing of Adrenal Aldosterone Producing Lesions Reveal Three Somatic Mutations near the KCNJ5 Potassium Channel Selectivity Filter

BackgroundAldosterone producing lesions are a common cause of hypertension, but genetic alterations for tumorigenesis have been unclear. Recently, either of two recurrent somatic missense mutations (G151R or L168R) was found in the potassium channel KCNJ5 gene in aldosterone producing adenomas. These mutations alter the channel selectivity filter and result in Na+ conductance and cell depolarization, stimulating aldosterone production and cell proliferation. Because a similar mutation occurs in a Mendelian form of primary aldosteronism, these mutations appear to be sufficient for cell proliferation and aldosterone production. The prevalence and spectrum of KCNJ5 mutations in different entities of adrenocortical lesions remain to be defined.Materials and MethodsThe coding region and flanking intronic segments of KCNJ5 were subjected to Sanger DNA sequencing in 351 aldosterone producing lesions, from patients with primary aldosteronism and 130 other adrenocortical lesions. The specimens had been collected from 10 different worldwide referral centers.ResultsG151R or L168R somatic mutations were identified in 47% of aldosterone producing adenomas, each with similar frequency. A previously unreported somatic mutation near the selectivity filter, E145Q, was observed twice. Somatic G151R or L168R mutations were also found in 40% of aldosterone producing adenomas associated with marked hyperplasia, but not in specimens with merely unilateral hyperplasia. Mutations were absent in 130 non-aldosterone secreting lesions. KCNJ5 mutations were overrepresented in aldosterone producing adenomas from female compared to male patients (63 vs. 24%). Males with KCNJ5 mutations were significantly younger than those without (45 vs. 54, respectively; p<0.005) and their APAs with KCNJ5 mutations were larger than those without (27.1 mm vs. 17.1 mm; p<0.005).DiscussionEither of two somatic KCNJ5 mutations are highly prevalent and specific for aldosterone producing lesions. These findings provide new insight into the pathogenesis of primary aldosteronism.

Sodium-Potassium ion Channel Selectivity can be Modeled by Thyrofluidic Channel Gating

Current views hold that the primary barrier to ion diffusion through a narrow pore is the energy required to dehydrate the transiting ion. The standard model for the selectivity filter of potassium channels consists of four carbonyls coordinating with the K+ ion, replacing the energy of ion hydration. Sodium ions, however, have diminished free energy due to their smaller radii, causing an increase in steric repulsion between the coordinating carbonyl groups. It has been recognized that this model does not completely account for experimentally measured selectivity, however.In the thyrofluidic ion channel model, the gating mechanism for a channel is the ion hydration state. A sufficient membrane electric field at the ion pore entrance will strip water molecules from the ion, facilitating ion entry. The electric field decays exponentially away from the membrane within several nanometers. If the ion channel extends too far from the membrane negligible hydration stripping will occur, and the hydration shell will remain around the ion, inhibiting entry. In addition to regulating channel gating, this mechanism also helps to account for the selectivity exhibited by Na+ and K+ channels. Our measurements of ion mobility in an electric field show that hydration stripping occurs at 400 V/cm, the field 6-7 nm from the membrane. Potassium channels tend to extend past this distance, where hydrated Na+ ion radii exceed hydrated K+ radii. As such, ion coordination energy transfer for ion flux would be significantly greater for Na+. Sodium channels tend to lie within this boundary, however, so they will be exposed primarily to dehydrated ions. As such, sterically unfavorable ion coordination becomes unnecessary to allow ion flux. Thus, the effects of membrane electric field hydration stripping and channel extension create differential ion states at channel entry sites.

Mapping the sequence of conformational changes underlying selectivity filter gating in the Kv11.1 potassium channel

The potassium channel selectivity filter both discriminates between K(+) and sodium ions and contributes to gating of ion flow. Static structures of conducting (open) and nonconducting (inactivated) conformations of this filter are known; however, the sequence of protein rearrangements that connect these two states is not. We show that closure of the selectivity filter gate in the human K(v)11.1 K(+) channel (also known as hERG, for ether-a-go-go-related gene), a key regulator of the rhythm of the heartbeat, is initiated by K(+) exit, followed in sequence by conformational rearrangements of the pore domain outer helix, extracellular turret region, voltage sensor domain, intracellular domains and pore domain inner helix. In contrast to the simple wave-like sequence of events proposed for opening of ligand-gated ion channels, a complex spatial and temporal sequence of widespread domain motions connect the open and inactivated states of the K(v)11.1 K(+) channel.

A Twist on Potassium Channel Gating

The selectivity filter of potassium channels, the narrowest segment of the pore, permits only potassium ions to diffuse through the pore. Clarke et al. (2010) now present 11 structures of an inwardly rectifying potassium channel, providing evidence that the selectivity filter functions in channel gating and conformational changes in the cytoplasmic domains correlate with pore opening.

High-Resolution Crystallographic Analysis of the Kcsa Gating Cycle from Cysteine-Trapped Open Channels

The K+ channel pore domain contains all the elements necessary to catalyze the selective permeation of K+ ions, in addition to regulate events underlying activation and inactivation gating. In KcsA, an inactivation process related to C-type inactivation in eukaryotic channels has been attributed to putative conformational changes at the selectivity filter (SF)[1]. Previously, we have provided crystallographic evidence for the conformational changes associated to C-type inactivation, albeit at relatively low-resolution [2]. Here, we have taken advantage of a cysteine-bridged locked open KcsA-mutant to study the structural changes at the selectivity filter when the activation gate (AG) is open and the filter transitions between its conductive and non-conductive conformations. We report the structures of KcsA for the non-inactivating mutantE71A at 2.1 A; the fully inactivated mutant Y82A at 2.32 A; and the non-inactivating mutant F103A at 2.64 A, where the allosteric coupling between the two gates (AG and SF) has been impaired. This set of high-resolution structures for different KcsA kinetic states represent a sharp improvement over the resolution of non-cysteine trapped mutants and will be interpreted in relation to their complementary functional characterization.1. Cordero-Morales, J.F., et al., Molecular determinants of gating at the potassium-channel selectivity filter. Nat Struct Mol Biol, 2006. 13(4): p. 311-8.2. Cuello, L.G., et al. (2008). Structural basis of K+ channel C-type inactivation: Crystal structure of KcsA in the Open/C-type inactivated conformation. 52nd Annual meeting of the Biophysical Society. Mini-symposium

Conformational Changes and Gating at the Selectivity Filter of Potassium Channels

The translocation of ions and water across cell membranes is a prerequisite for many of life's processes. K(+) channels are a diverse family of integral membrane proteins through which K(+) can pass selectively. There is an ongoing debate about the nature of conformational changes associated with the opening and closing and conductive and nonconductive states of potassium (K(+)) channels. These changes depend on the membrane potential, the K(+) concentration gradient, and large scale motions of transmembrane helices and associated residues. Experiments also suggest that local structural changes in the selectivity filter may act as the dominant gate referred to as C-type inactivation. Herein we present an extensive computational study on KirBac, which supports the existence of a physical gate or constriction in the selectivity filter (SF) of K(+) channels. Our computations identify a new selectivity filter structure, which is likely associated with C-type inactivation. Specifically, the four peptide chains that comprise the filter adopt an unusual structure in which their dihedrals alternate between left- and right-handed Ramachandran angles, which also justifies the need for conservation of glycine in the K(+) selectivity filter, since it is the only residue able to play this bifunctional role.

The Molecular Mechanism of Toxin-Induced Conformational Changes in a Potassium Channel: Relation to C-Type Inactivation

Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.

K + Conduction in the Selectivity Filter of Potassium Channels Is Monitored by the Charge Distribution along Their Sequence

Potassium channels display a high conservation of sequence of the selectivity filter (SF), yet nature has designed a variety of channels that present a wide range of absolute rates of K + permeation. In KcsA, the structural archetype for K channels, under physiological concentrations, two K + ions reside in the SF in configurations 1,3 (up state) and 2,4 (down state) and ion conduction is believed to follow a throughput cycle involving a transition between these states. Using free-energy calculations of KcsA, Kv1.2, and mutant channels, we show that this transition is characterized by a channel-dependent energy barrier. This barrier is strongly influenced by the charges partitioned along the sequence of each channel. These results unveil therefore how, for similar structures of the SF, the rate of K + turnover may be fine-tuned within the family of potassium channels.

Ion pore properties of ionotropic glutamate receptors are modulated by a transplanted potassium channel selectivity filter

The canonical potassium channel selectivity filter motif TVGYG was transplanted into ionotropic glutamate receptors (iGluRs) of the AMPA and NMDA subtype to test whether it renders the iGluRs K + selective. The TVGYG motif modulated several ion pore properties of AMPA receptor as well as NMDA receptor mutants, e.g., the intra- and extracellular polyamine block, current/voltage relationships, open channel block by MK801 and Mg 2+, and permeability for divalent cations. However, introduction of the selectivity filter failed to increase the K + selectivity of homomeric AMPA and heteromeric NMDA receptor complexes, which may be due to absence of selectivity filter-stabilizing interaction sites in the iGluR pore domain. Our findings indicate that even if glutamate receptors appear to have the intrinsic capacity for K + permeability, as is demonstrated by the prokaryotic, glutamate-gated, K + selective GluR0, the isolated selectivity filter is not able to confer K + permeability to the relatively unselective iGluR cation pore.

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 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 Pore Helix Dipole Has a Minor Role in Inward Rectifier Channel Function

Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.

A Gate in the Selectivity Filter of Potassium Channels

The selectivity filter of potassium channels is the structural element directly responsible for the selective and rapid conduction of K+, whereas other parts of the protein are thought to function as a molecular gate that either permits or blocks the passage of ions. However, whether the selectivity filter itself also possesses the ability to play the role of a gate is an unresolved question. Using free energy molecular dynamics simulations, it is shown that the reorientation of two peptide linkages in the selectivity filter of the KcsA K+ channel can lead to a stable nonconducting conformational state. Two microscopic factors influence the transition toward such a conformational state: the occupancy of one specific cation binding site in the selectivity filter (S2), and the strength of intersubunit interactions involving the GYG signature sequence. These results suggest that such conformational transitions occurring in the selectivity filter might be related to different K+ channel gating events, including C-type (slow) inactivation.

Hierarchy of Motions and Quasi-Particles in a Simplified Model of Potassium Channel Selectivity Filter

We develop a simplified model of themultiply occupied Kcsa-like selectivityfilter based on the best availablestructural data. The existence of hierarchyof motions in the selectivity filter isshown. Fast fluctuations of the ion-iondistances may be considered adiabaticallydecoupled from the slow diffusive motion ofthe ions' center of masses. The latter canbe considered as a quasi-particle, called aquasi-ion, moving in an effectivepotential. In the Kcsa-like selectivityfilter occupied by three ions the effectivepotential allows free barrier-lessdiffusional motion of the quasi-ions. Theconcept of the quasi-ions performing iontranslocation through the channel may bevital in explaining barrier-less `knock-on' conduction postulated for real channels.

The Occupancy of Ions in the K + Selectivity Filter: Charge Balance and Coupling of Ion Binding to a Protein Conformational Change Underlie High Conduction Rates

Potassium ions diffuse across the cell membrane in a single file through the narrow selectivity filter of potassium channels. The crystal structure of the KcsA K + channel revealed the chemical structure of the selectivity filter, which contains four binding sites for K +. In this study, we used Tl + in place of K + to address the question of how many ions bind within the filter at a given time, i.e. what is the absolute ion occupancy? By refining the Tl + structure against data to 1.9 Å resolution with an anomalous signal, we determined the absolute occupancy of Tl +. Then, by comparing the electron density of Tl + with that of K +, Rb + and Cs +, we estimated the absolute occupancy of these three ions. We further analyzed how the ion occupancy affects the conformation of the selectivity filter by analyzing the structure of KcsA at different concentrations of Tl +. Our results indicate that the average occupancy for each site in the selectivity filter is about 0.63 for Tl + and 0.53 for K +. For K +, Rb + and Cs +, the total number of ions contained within four sites in the selectivity filter is about two. At low concentrations of permeant ion, the number of ions drops to one in association with a conformational change in the selectivity filter. We conclude that electrostatic balance and coupling of ion binding to a protein conformational change underlie high conduction rates in the setting of high selectivity.

Interchain hydrogen-bonding interactions may facilitate translocation of K+ ions across the potassium channel selectivity filter, as suggested by synthetic modeling chemistry.

A 4-fold symmetric arrangement of TVGYG polypeptides forms the selectivity filter of the K+ channel from Streptomyces lividans (KcsA). We report the synthesis and properties of synthetic models for the filter, p-tert-butyl-calix[4]arene-(OCH(2)CO-XOBz)(4) (X = V, VG, VGY), 1-3. The first cation (Na+, K+) binds to the four -[OCH(2)CO]- units, a region devised to mimic the metal-binding site formed by the four T residues in KcsA. NMR studies reveal that cations and valine amide protons compete for the carbonyl oxygen atoms, converting NH(Val)...O=C hydrogen bonds to M+ ...O=C bonds (M+ = Na+ or K+). The strength of these interchain NH(Val)...O=C hydrogen bonds varies in the order 3 > 2 > 1. We propose that such interchain H-bonding may destabilize metal binding in the selectivity filter and thus help create the low energy barrier needed for rapid cation translocation.

Modulation of C-Type Inactivation by K + at the Potassium Channel Selectivity Filter

With prolonged or repetitive activation, voltage-gated K + channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the rate at which K + leaves an unidentified binding site in the pore. We have functionally identified two K + binding sites in the conduction pathway of a chimeric K + channel that conducts Na + in the absence of K +. One site has a high affinity for K + and contributes to the selectivity filter mechanism for K + over Na +. Another site, external to the high-affinity site, has a lower affinity for K + and is not involved in channel selectivity. Binding of K + to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K + at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K +.