Exploring the Viral Channel KcvPBCV-1 Function via Computation.
ABSTRACT: Viral potassium channels (Kcv) are homologous to the pore module of complex [Formula: see text]-selective ion channels of cellular organisms. Due to their relative simplicity, they have attracted interest towards understanding the principles of [Formula: see text] conduction and channel gating. In this work, we construct a homology model of the [Formula: see text] open state, which we validate by studying the binding of known blockers and by monitoring ion conduction through the channel. Molecular dynamics simulations of this model reveal that the re-orientation of selectivity filter carbonyl groups coincides with the transport of potassium ions, suggesting a possible mechanism for fast gating. In addition, we show that the voltage sensitivity of this mechanism can originate from the relocation of potassium ions inside the selectivity filter. We also explore the interaction of [Formula: see text] with the surrounding bilayer and observe the binding of lipids in the area between two adjacent subunits. The model is available to the scientific community to further explore the structure/function relationship of Kcv channels.
Project description:In the quest for developing novel and efficient batteries, a great interest has been raised for sustainable K-based honeycomb layer oxide materials, both for their application in energy devices as well as for their fundamental material properties. A key issue in the realization of efficient batteries based on such compounds, is to understand the K-ion diffusion mechanism. However, investigation of potassium-ion (K[Formula: see text]) dynamics in materials using e.g. NMR and related techniques has so far been very challenging, due to its inherently weak nuclear magnetic moment, in contrast to other alkali ions such as lithium and sodium. Spin-polarised muons, having a high gyromagnetic ratio, make the muon spin rotation and relaxation ([Formula: see text]SR) technique ideal for probing ions dynamics in these types of energy materials. Here we present a study of the low-temperature magnetic properties as well as K[Formula: see text] dynamics in honeycomb layered oxide material [Formula: see text] using mainly the [Formula: see text]SR technique. Our low-temperature [Formula: see text]SR results together with complementary magnetic susceptibility measurements find an antiferromagnetic transition at [Formula: see text] K. Further [Formula: see text]SR studies performed at higher temperatures reveal that potassium ions (K[Formula: see text]) become mobile above 200 K and the activation energy for the diffusion process is obtained as [Formula: see text] meV. This is the first time that K[Formula: see text] dynamics in potassium-based battery materials has been measured using [Formula: see text]SR. Assisted by high-resolution neutron diffraction, the temperature dependence of the K-ion self diffusion constant is also extracted. Finally our results also reveal that K-ion diffusion occurs predominantly at the surface of the powder particles. This opens future possibilities for potentially improving ion diffusion as well as K-ion battery device performance using nano-structuring and surface coatings of the particles.
Project description:Chlorella virus PBCV-1 (Paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In this paper, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisiae mutants that lack endogenous K+ uptake systems. Compared with the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, barium and caesium. Single channel analyses reveal a stochastic gating behaviour and a voltage-dependent conductance that resembles the macroscopic I/V relationship. One difference between PBCV-1 and ATCV-1 Kcv is that the latter is more permeable to K+ than Rb+. This difference is partially explained by the presence of a tyrosine residue in the selective filter of ATCV-1 Kcv, whereas PBCV-1 Kcv has a phenylalanine. Hence, ATCV-1 Kcv is the smallest protein to form a K+ channel and it will serve as a model for studying structure-function correlations inside the potassium channel pore.
Project description:Potassium channels modulate various cellular functions through efficient and selective conduction of K<sup>+</sup> ions. The mechanism of ion conduction in potassium channels has recently emerged as a topic of debate. Crystal structures of potassium channels show four K<sup>+</sup> ions bound to adjacent binding sites in the selectivity filter, while chemical intuition and molecular modeling suggest that the direct ion contacts are unstable. Molecular dynamics (MD) simulations have been instrumental in the study of conduction and gating mechanisms of ion channels. Based on MD simulations, two hypotheses have been proposed, in which the four-ion configuration is an artifact due to either averaged structures or low temperature in crystallographic experiments. The two hypotheses have been supported or challenged by different experiments. Here, MD simulations with polarizable force fields validated by <i>ab initio</i> calculations were used to investigate the ion binding thermodynamics. Contrary to previous beliefs, the four-ion configuration was predicted to be thermodynamically stable after accounting for the complex electrostatic interactions and dielectric screening. Polarization plays a critical role in the thermodynamic stabilities. As a result, the ion conduction likely operates through a simple single-vacancy and water-free mechanism. The simulations explained crystal structures, ion binding experiments and recent controversial mutagenesis experiments. This work provides a clear view of the mechanism underlying the efficient ion conduction and demonstrates the importance of polarization in ion channel simulations.
Project description:Kcv, a 94-aa protein encoded by Paramecium bursaria chlorella virus 1, is the smallest known protein to form a functional potassium ion channel and basically corresponds to the "pore module" of potassium channels. Both viral replication and channel activity are inhibited by the ion channel blockers barium and amantadine but not by cesium. Genes encoding Kcv-like proteins were isolated from 40 additional chlorella viruses. Differences in 16 of the 94 amino acids were detected, producing six Kcv-like proteins with amino acid substitutions occurring in most of the functional domains of the protein (N terminus, transmembrane 1, pore helix, selectivity filter, and transmembrane 2). The six proteins form functional potassium selective channels in Xenopus oocytes with different properties including altered current kinetics and inhibition by cesium. The amino acid changes together with the different properties observed in the six Kcv-like channels will be used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv.
Project description:Voltage-gated potassium channels open at depolarized membrane voltages. A prolonged depolarization causes a rearrangement of the selectivity filter which terminates the conduction of ions - a process called slow or C-type inactivation. How structural rearrangements in the voltage-sensor domain (VSD) cause alteration in the selectivity filter, and vice versa, are not fully understood. We show that pulling the pore domain of the Shaker potassium channel towards the VSD by a Cd(2+) bridge accelerates C-type inactivation. Molecular dynamics simulations show that such pulling widens the selectivity filter and disrupts the K(+) coordination, a hallmark for C-type inactivation. An engineered Cd(2+) bridge within the VSD also affect C-type inactivation. Conversely, a pore domain mutation affects VSD gating-charge movement. Finally, C-type inactivation is caused by the concerted action of distant amino acid residues in the pore domain. All together, these data suggest a reciprocal communication between the pore domain and the VSD in the extracellular portion of the channel.
Project description:Although extensively studied, it has proved difficult to describe in detail how potassium ion channels conduct cations and water. We present a computational study that, by using stratified umbrella sampling, examines nearly an entire conduction event of the Kv1.2/2.1 paddle chimera and thereby identifies the expected stable configurations of ions and waters in the selectivity filter of the channel. We describe in detail the motions of the ions and waters during a conduction event, focusing on how waters and ions enter the filter, the rotation of water molecules inside the filter, and how potassium ions are coordinated as they move from a water to a protein environment. Finally, we analyze the small conformational changes undergone by the protein, showing that the stable configurations are most similar to the experimental crystal structure.
Project description:<h4>Background</h4>The pyrethroid deltamethrin (DM) is broadly used for insect control. Although DM hyperexcites neuronal networks by delaying inactivation of axonal voltage-dependent [Formula: see text] channels, this mechanism is unlikely to mediate neurotoxicity at lower exposure levels during critical perinatal periods in mammals.<h4>Objectives</h4>We aimed to identify mechanisms by which acute and subchronic DM altered axonal and dendritic growth, patterns of synchronous [Formula: see text] oscillations (SCOs), and electrical spike activity (ESA) functions critical to neuronal network formation.<h4>Methods</h4>Measurements of SCOs using [Formula: see text] imaging, ESA using microelectrode array (MEA) technology, and dendritic complexity using Sholl analysis were performed in primary murine cortical neurons from wild-type (WT) and/or ryanodine receptor 1 ([Formula: see text]) mice between 5 and 14 d in vitro (DIV). [Formula: see text] binding analysis and a single-channel voltage clamp were utilized to measure engagement of RyRs as a direct target of DM.<h4>Results</h4>Neuronal networks responded to DM ([Formula: see text]) as early as 5 DIV, reducing SCO amplitude and depressing ESA and burst frequencies by 60-70%. DM ([Formula: see text]) enhanced axonal growth in a nonmonotonic manner. [Formula: see text] enhanced dendritic complexity. DM stabilized channel open states of RyR1, RyR2, and cortical preparations expressing all three isoforms. DM ([Formula: see text]) altered gating kinetics of RyR1 channels, increasing mean open time, decreasing mean closed time, and thereby enhancing overall open probability. SCO patterns from cortical networks expressing [Formula: see text] were more responsive to DM than WT. [Formula: see text] neurons showed inherently longer axonal lengths than WT neurons and maintained less length-promoting responses to nanomolar DM.<h4>Conclusions</h4>Our findings suggested that RyRs were sensitive molecular targets of DM with functional consequences likely relevant for mediating abnormal neuronal network connectivity in vitro. https://doi.org/10.1289/EHP4583.
Project description:Potassium channels can conduct passively K+ ions with rates of up to approximately 10(8) ions per second at physiological conditions, and they are selective to these species by a factor of 10(4) over Na+ ions. Ion conduction has been proposed to involve transitions between 2 main states, with 2 or 3 K+ ions occupying the selectivity filter separated by an intervening water molecule. The largest free energy barrier of such a process was reported to be of the order of 2-3 kcal mol(-1). Here, we present an alternative mechanism for conduction of K+ in potassium channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. Conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of K+-water-K+.
Project description:Generation of action potentials (APs) is a crucial step in neuronal information processing. Existing biophysical models for AP generation almost universally assume that individual voltage-gated sodium channels operate statistically independently, and their avalanche-like opening that underlies AP generation is coordinated only through the transmembrane potential. However, biological ion channels of various types can exhibit strongly cooperative gating when clustered. Cooperative gating of sodium channels has been suggested to explain rapid onset dynamics and large threshold variability of APs in cortical neurons. It remains however unknown whether these characteristic properties of cortical APs can be reproduced if only a fraction of channels express cooperativity, and whether the presence of cooperative channels has an impact on encoding properties of neuronal populations. To address these questions we have constructed a conductance-based neuron model in which we continuously varied the size of a fraction [Formula: see text] of sodium channels expressing cooperativity and the strength of coupling between cooperative channels [Formula: see text]. We show that starting at a critical value of the coupling strength [Formula: see text], the activation curve of sodium channels develops a discontinuity at which opening of all coupled channels becomes an all-or-none event, leading to very rapid AP onsets. Models with a small fraction, [Formula: see text], of strongly cooperative channels generate APs with the most rapid onset dynamics. In this regime APs are triggered by simultaneous opening of the cooperative channel fraction and exhibit a pronounced biphasic waveform often observed in cortical neurons. We further show that presence of a small fraction of cooperative Na+ channels significantly improves the ability of neuronal populations to phase-lock their firing to high frequency input fluctuation. We conclude that presence of a small fraction of strongly coupled sodium channels can explain characteristic features of cortical APs and has a functional impact of enhancing the spike encoding of rapidly varying signals.
Project description:Proteins that form ion-selective pores in the membrane of cells are integral to many rapid signaling processes, including regulating the rhythm of the heartbeat. In potassium channels, the selectivity filter is critical for both endowing an exquisite selectivity for potassium ions, as well as for controlling the flow of ions through the pore. Subtle rearrangements in the complex hydrogen-bond network that link the selectivity filter to the surrounding pore helices differentiate conducting (open) from nonconducting (inactivated) conformations of the channel. Recent studies suggest that beyond the selectivity filter, inactivation involves widespread rearrangements of the channel protein. Here, we use rate equilibrium free energy relationship analysis to probe the structural changes that occur during selectivity filter gating in Kv11.1 channels, at near atomic resolution. We show that the pore helix plays a crucial dynamic role as a bidirectional interface during selectivity filter gating. We also define the molecular bases of the energetic coupling between the pore helix and outer helix of the pore domain that occurs early in the transition from open to inactivated states, as well as the coupling between the pore helix and inner helix late in the transition. Our data demonstrate that the pore helices are more than just static structural elements supporting the integrity of the selectivity filter; instead they play a crucial dynamic role during selectivity filter gating.