K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study.
ABSTRACT: Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.
Project description:The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski's Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na(+) and K(+) ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na(+) and K(+). These structural rearrangements facilitate entry of K(+) ions into the selectivity filter and permeation through the channel, and rejection of Na(+) ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K(+). Estimates of the K(+)/Na(+) selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na(+) ions, the "punch through" relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.
Project description:Potassium channels enable K(+) ions to move passively across biological membranes. Multiple nanosecond-duration molecular dynamics simulations (total simulation time 5 ns) of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal motions of ions, water, and protein. Comparison of simulations with and without K(+) ions indicate that the absence of ions destabilizes the structure of the selectivity filter. Within the selectivity filter, K(+) ions interact with the backbone (carbonyl) oxygens, and with the side-chain oxygen of T75. Concerted single-file motions of water molecules and K(+) ions within the selectivity filter of the channel occur on a 100-ps time scale. In a simulation with three K(+) ions (initially two in the filter and one in the cavity), the ion within the central cavity leaves the channel via its intracellular mouth after approximately 900 ps; within the cavity this ion interacts with the Ogamma atoms of two T107 side chains, revealing a favorable site within the otherwise hydrophobically lined cavity. Exit of this ion from the channel is enabled by a transient increase in the diameter of the intracellular mouth. Such "breathing" motions may form the molecular basis of channel gating.
Project description:An understanding of ion-protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion-protein interactions in the KcsA selectivity filter. A band at 1680 cm-1 in the amide I region would be a marker band for the ion occupancy of K+, Rb+, and Cs+ in the filter. The band at 1627 cm-1 observed in both Na+ and Li+ conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K+ ions provides quantitative information on the ion affinity of the selectivity filter.
Project description:The bacterial channel KirBac1.1 provides a structural homolog of mammalian inward rectifier potassium (Kir) channels. The conformational dynamics of the selectivity filter of Kir channels are of some interest in the context of possible permeation and gating mechanisms for this channel. Molecular dynamics simulations of KirBac have been performed on a 10-ns timescale, i.e., comparable to that of ion permeation. The results of five simulations (total simulation time 50 ns) based on three different initial ion configurations and two different model membranes are reported. These simulation data provide evidence for limited (<0.1 nm) filter flexibility during the concerted motion of ions and water molecules within the filter, such local changes in conformation occurring on an approximately 1-ns timescale. In the absence of K(+) ions, the KirBac selectivity filter undergoes more substantial distortions. These resemble those seen in comparable simulations of other channels (e.g., KcsA and KcsA-based homology models) and are likely to lead to functional closure of the channel. This suggests filter distortions may provide a mechanism of K-channel gating in addition to changes in the hydrophobic gate formed at the intracellular crossing point of the M2 helices. The simulation data also provide evidence for interactions of the "slide" (pre-M1) helix of KirBac with phospholipid headgroups.
Project description:Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.
Project description:Atomic resolution structures have provided significant insight into the gating and permeation mechanisms of various ion channels, including potassium channels. However, ion channels may also be regulated by numerous factors, including the physiochemical properties of the membrane in which they are embedded. For example, the matching of the bilayer's hydrophobic region to the hydrophobic external surface of the ion channel is thought to minimize the energetic penalty needed to solvate hydrophobic residues or exposed lipid tails. To understand the molecular basis of such regulation by hydrophobic matching requires examining channels in the presence of the lipid membrane. Here we examine the role of hydrophobic matching in regulating the activity of the model potassium channel, KcsA. 86Rb+ influx assays and single-channel recordings indicate that the non-inactivating E71A KcsA channel is most active in thin bilayers (<diC18:1PC). Bilayer thickness affects the open probability of KcsA and not its unitary conductance. Molecular dynamics simulations indicate that the bilayer can sufficiently modify its dimensions to accommodate KcsA channels without major perturbations in the protein helical packing within the nanosecond timescale. Based on experimental results and MD simulations, we present a model in which bilayer thickness influences the stability of the open and closed conformations of the intracellular gate of KcsA, with minimal impact on the stability of the selectivity filter of the non-inactivating mutant, E71A.
Project description:The mechanism by which K(+) channels select for K(+) over Na(+) ions has been debated for the better part of a century. The prevailing view is that K(+) channels contain highly conserved sites that selectively bind K(+) over Na(+) ions through optimal coordination. We demonstrate that a series of alternating sites within the KcsA channel selectivity filter exists, which are thermodynamically selective for either K(+) (cage made from two planes of oxygen atoms) or Na(+) ions (a single plane of four oxygen atoms). By combining Bennett free energy perturbation calculations with umbrella sampling, we show that when K(+) and Na(+) are both permitted to move into their preferred positions, the thermodynamic preference for K(+) over Na(+) is significantly reduced throughout the entire selectivity filter. We offer a rationale for experimental measures of thermodynamic preference for K(+) over Na(+) from Ba(2+) blocking data, by demonstrating that the presence of Ba(2+) ions exaggerates K(+) over Na(+) thermodynamic stability due to the different binding locations of these ions. These studies reveal that K(+) channel selectivity may not be associated with the thermodynamics of ions in crystallographic K(+) binding sites, but requires consideration of the kinetic barriers associated with the different multi-ion permeation mechanisms.
Project description:K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K(+) channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na(+) or low [K(+)]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K(+) ion binding to channels with an inactivated or conductive selectivity filter is different from K(+) ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.
Project description: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 A in closed KcsA (Calpha-Calpha distances at Thr 112) to 32 A 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.
Project description:A universal property of ion channels is their ability to alternate stochastically between two permeation states, open and closed. This behavior is thought to be controlled by a steric "gate", a structure that physically impedes ion flow in the closed state and moves out of the way during channel opening. Experiments employing macroscopic currents in the Shaker K channel have suggested a cytoplasmic localization for the gate. Crystallographic structures of the KcsA K channel indeed reveal a cytoplasmic constriction, implying that the gate and selectivity filter are localized to opposite ends of the permeation pathway. However, analysis of K channel subconductance behavior has suggested a strict coupling between channel opening (gating) and permeation. The idea that the selectivity filter is the gate was therefore investigated by using Monte Carlo simulations. Gating is accomplished by allowing the filter to alternate stochastically between two conformations: a high-affinity state, which selectively binds K ions (but not Na ions) and traps them, and a completely nonselective, low-affinity state, which allows both Na and K ions to permeate. The results of these simulations indicate that affinity switching not only endows the selectivity filter with gating abilities, it also allows efficient permeation without jeopardizing ion selectivity. In this model, permeation and gating result from the same process.