Exploration of the pore structure of a peptide-gated Na+ channel

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.     

K(+)/Na(+) selectivity of the KcsA potassium channel from microscopic free energy perturbation calculations

Microscopic molecular dynamics free energy perturbation calculations of the K(+)/Na(+) selectivity in the KcsA potassium channel, based on its experimental three-dimensional structure, are reported. The relative binding free energies for K(+) and Na(+) in the most relevant ion occupancy states of the four-site selectivity filter are calculated. The previously proposed mechanism for ion permeation through the KcsA channel is predicted, in agreement with available experimental data, to have a significant selectivity for K(+) over Na(+). The calculations also show that the individual 'binding site' selectivities are generally not additive and the doubly loaded states of the filter thus display cooperative effects. The only site that is not K(+) selective is that which is located at the entrance to the internal water cavity, suggesting the possibility that internal Na(+) could block outward currents.     

Molecular dynamics of the KcsA K(+) channel in a bilayer membrane

Molecular dynamics (MD) simulations of an atomic model of the KcsA K(+) channel embedded in an explicit dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer solvated by a 150 mM KCl aqueous salt solution are performed and analyzed. The model includes the KcsA K(+) channel, based on the recent crystallographic structure of, Science. 280:69-77), 112 DPPC, K(+) and Cl(-) ions, as well as over 6500 water molecules for a total of more than 40,000 atoms. Three K(+) ions are explicitly included in the pore. Two are positioned in the selectivity filter on the extracellular side and one in the large water-filled cavity. Different starting configurations of the ions and water molecules in the selectivity filter are considered, and MD trajectories are generated for more than 4 ns. The conformation of KcsA is very stable in all of the trajectories, with a global backbone root mean square (RMS) deviation of less than 1.9 A with respect to the crystallographic structure. The RMS atomic fluctuations of the residues surrounding the selectivity filter on the extracellular side of the channel are significantly lower than those on the intracellular side. The motion of the residues with aromatic side chains surrounding the selectivity filter (Trp(67), Trp(68), Tyr(78), and Tyr(82)) is anisotropic with the smallest RMS fluctuations in the direction parallel to the membrane plane. A concerted dynamic transition of the three K(+) ions in the pore is observed, during which the K(+) ion located initially in the cavity moves into the narrow part of the selectivity filter, while the other two K(+) ions move toward the extracellular side. A single water molecule is stabilized between each pair of ions during the transition, suggesting that each K(+) cation translocating through the narrow pore is accompanied by exactly one water molecule, in accord with streaming potential measurements (, Biophys. J. 55:367-371). The displacement of the ions is coupled with the structural fluctuations of Val(76) and Gly(77), in the selectivity filter, as well as the side chains of Glu(71), Asp(80), and Arg(89), near the extracellular side. Thus the mechanical response of the channel structure at distances as large as 10-20 A from the ions in the selectivity filter appears to play an important role in the concerted transition.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel

TEA is a classical blocker of K(+) channels. From mutagenesis studies, it has been shown that external blockade by TEA is strongly dependent upon the presence of aromatic residue at Shaker position 449 which is located near the extracellular entrance to the pore (Heginbotham, L., and R. MacKinnon. 1992. Neuron. 8:483-491). The data suggest that TEA interacts simultaneously with the aromatic residues of the four monomers. The determination of the 3-D structure of the KcsA channel using X-ray crystallography (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) has raised some issues that remain currently unresolved concerning the interpretation of these observations. In particular, the center of the Tyr82 side chains in KcsA (corresponding to position 449 in Shaker) forms a square of 11.8-A side, a distance which is too large to allow simultaneous interactions of a TEA molecule with the four aromatic side chains. In this paper, the external blockade by TEA is explored by molecular dynamics simulations of an atomic model of KcsA in an explicit phospholipid bilayer with aqueous salt solution. It is observed, in qualitative accord with the experimental results, that TEA is stable when bound to the external side of the wild-type KcsA channel (with Tyr82), but is unstable when bound to a mutant channel in which the tyrosine residue has been substituted by a threonine. The free energy profile of TEA relative to the pore is calculated using umbrella sampling simulations to characterize quantitatively the extracellular blockade. It is found, in remarkable agreement with the experiment, that the TEA is more stably bound by 2.3 kcal/mol to the channel with four tyrosine residues. In the case of the wild-type KcsA channel, TEA (which has the shape of a flattened oblate spheroid) acts as an ideal plug blocking the pore. In contrast, it is considerably more off-centered and tilted in the case of the mutant channel. The enhanced stability conferred by the tyrosine residues does not arise from Pi-cation interactions, but appears to be due to differences in the hydration structure of the TEA. Finally, it is shown that the experimentally observed voltage dependence of TEA block, which is traditionally interpreted in terms of the physical position of the TEA along the axis of the pore, must arise indirectly via coupling with the ions in the pore.     

Functional influence of the pore helix glutamate in the KcsA K+ channel

The E71 residue is buried near the selectivity filter in the KcsA K+ channel and forms a carboxyl-carboxylate bridge with D80. We have investigated the importance of E71 by examining neutralization mutants at this position using biochemical and electrophysiological methods. E71 mutations differentially destabilize the detergent-solubilized tetramer; among them, the E71V neutralization mutant has a relatively subtle effect. The E71V channel displays electrical activity when reconstituted into planar lipid bilayers. In single channel recordings, the mutant retains K+/Na+ selectivity, and its conductance in the outward direction is unaltered. Some conduction properties are changed: inward conductance is increased. Our results show that that the E71 side chain is not a primary determinant of ion selectivity or conduction in the wild-type channel, either directly or through the E71:D80 carboxyl-carboxylate bridge.     

Energetics of pore opening in a voltage-gated K(+) channel

Voltage-dependent gating in K(+) channels results from the mechanical coupling of voltage sensor movements to pore opening. We used single and double mutations in the pore of the Shaker K(+) channel to analyze a late concerted pore opening transition and interpreted the results in the context of known K(+) channel structures. Gating sensitive mutations are located at mechanistically informative regions of the pore and are coupled energetically across distances up to 15 A. We propose that the pore is intrinsically more stable when closed, and that to open the pore the voltage sensors must exert positive work by applying an outward lateral force near the inner helix bundle.     

The interaction of Na(+) and K(+) in the pore of cyclic nucleotide-gated channels

The permeability ratio between K(+) and Na(+) ions in cyclic nucleotide-gated channels is close to 1, and the single channel conductance has almost the same value in the presence of K(+) or Na(+). Therefore, K(+) and Na(+) ions are thought to permeate with identical properties. In the alpha-subunit from bovine rods there is a loop of three prolines at positions 365 to 367. When proline 365 is mutated to a threonine, a cysteine, or an alanine, mutant channels exhibit a complex interaction between K(+) and Na(+) ions. Indeed K(+), Rb(+) and Cs(+) ions do not carry any significant macroscopic current through mutant channels P365T, P365C and P365A and block the current carried by Na(+) ions. Moreover in mutant P365T the presence of K(+) in the intracellular (or extracellular) medium caused the appearance of a large transient inward (or outward) current carried by Na(+) when the voltage command was quickly stepped to large negative (or positive) membrane voltages. This transient current is caused by a transient potentiation, i.e., an increase of the open probability. The permeation of organic cations through these mutant channels is almost identical to that through the wild type (w.t.) channel. Also in the w.t. channel a similar but smaller transient current is observed, associated to a slowing down of the channel gating evident when intracellular Na(+) is replaced with K(+). As a consequence, a rather simple mechanism can explain the complex behavior here described: when a K(+) ion is occupying the pore there is a profound blockage of the channel and a potentiation of gating immediately after the K(+) ion is driven out. Potentiation occurs because K(+) ions slow down the rate constant K(off) controlling channel closure. These results indicate that K(+) and Na(+) ions do not permeate through CNG channels in the same way and that K(+) ions influence the channel gating.     

Global twisting motion of single molecular KcsA potassium channel upon gating

Ion channels are signal transduction molecules that switch ion permeation pathways on and off (gating). Crystal structures of several kinds of potassium channels have revealed open and closed conformations, which provide static pictures of gating status. Here we studied KcsA potassium channels undergoing conformational changes at the single-molecule level. A KcsA channel with a gold nanocrystal attached was irradiated by white X-rays and motions of the diffraction spot from the nanocrystal were tracked in real time. Upon gating, the KcsA channels twisted around the axis of the pore. These conformational changes were prevented by an open-channel blocker, tetrabuthylammonium. Random clockwise and counterclockwise twisting in the range of several tens of degrees originated in the transmembrane domain and was transmitted to the cytoplasmic domain. This coupling suggests a mechanical interplay between the transmembrane and cytoplasmic domains.     

Rho small GTPases activate the epithelial Na(+) channel

Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na(+) channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P(2) levels promoting channel insertion into the plasma membrane.     

Accelerated diffusion of Na(+) in a hydrophobic region revealed by molecular dynamics simulations of a synthetic ion channel

To get insight into the significance of the hydrophobic lining on the ion permeation, we performed molecular dynamics simulations on a Na(+) permeation through a de novo synthetic hydrophobic channel. Electrophysiological study has suggested that the channel is formed from a tail-to-tail associated dimer of a cyclic octa-peptide coupled with hydrophobic acyl chains. The acyl chains line the channel pore while the cyclic peptide forms the channel entrance [Z. Qi, M. Sokabe, K. Donowaki, H. Ishida, Biophys. J. 76 (1999) 631]. Molecular dynamics simulation of water in the channel indicated that the inferred structure is physically reasonable [Z. Qi, M. Sokabe, Biophys. Chem. 71 (1998) 35]. In the present study, the potential energy profile of the Na(+) and the energy contributions from each component of the system at different positions along the channel axis were calculated. An energy well instead of a peak is located at the central hydrophobic cavity of the channel, due to its ability of accommodating at least five water molecules to hydrate the ion. Interestingly, the ion diffuses much faster in the hydrophobic acyl chain region, particularly in the central hydrophobic cavity, than it does in the peptide ring region and even surprisingly faster than that in the bulk phase. These results provide a physical basis for an idea that the hydrophobic lining of the K(+) channel [D.A. Doyle, J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, R. MacKinnon, Science 280 (1998) 69] plays an active role to facilitate the ion permeation through the channel pore.     

The ionization state and the conformation of Glu-71 in the KcsA K(+) channel.

The side chain of Glu-71 of the KcsA K(+) channel, an important residue in the vicinity of the selectivity filter, was not resolved in the crystallographic structure of Doyle et al. (Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. Science. 280:69-77). Its atomic coordinates are undetermined and its ionization state is unknown. For meaningful theoretical and computational studies of the KcsA K(+) channel, it is essential to address questions about the conformation and the ionization state of this residue in detail. In previous MD simulations in which the side chain of Glu-71 is protonated and forming a strong hydrogen bond with Asp-80 it was observed that the channel did not deviate significantly from the crystallographic structure (Bernèche, S., and B. Roux. 2000. Biophys. J. 78:2900-2917). In contrast, we show here that the structure of the selectivity filter of the KcsA channel is significantly disrupted when these side chains are fully ionized on each of the four monomers. To further resolve questions about the ionization state of Glu-71 we calculated the pK(a) value of this residue using molecular dynamics free energy simulations (MD/FES) with a fully flexible system including explicit solvent and membrane and finite-difference Poisson-Boltzmann (PB) continuum electrostatics. It is found that the pK(a) of Glu-71 is shifted by approximately +10 pK(a) units. These results strongly suggest that Glu-71 is protonated under normal conditions.     

A molecular model of the inner pore of the Ca channel in its open state

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel¹ that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of ≠-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.     

A molecular model of the inner pore of the Ca channel in its open state

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel¹ that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of π-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.Key words: calcium channels, homology modeling, π-bulges, restrained minimization     

Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Clustering and coupled gating modulate the activity in KcsA, a potassium channel model

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na(+), and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na(+) blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

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.     

Self-electroporation as a model for fusion pore formation

The creation of a small opening called the fusion pore is a necessary prerequisite for neurotransmitter release from synaptic vesicles. It is known that high intensity electric fields can create pores in vesicles by a process called electroporation. Due to the presence of charged phosphatidylserine (PS) molecules on the inner leaflet of the cell membrane, an electric field that is strong enough to cause electroporation of a synaptic vesicle might be present. It was shown by K. Rosenheck [K. Rosenheck. Biophys J 75, 1237-1243 (1998)] that in a planar geometry, fields sufficient to cause electroporation can occur at intermembrane separations of less than approximately 3 nm. It is frequently found, however, that the cell membrane is not planar but caves inward at the locations where a vesicle is close to it. Indentation of the cell membrane in the fusion region was modelled as a hemisphere and a theoretical study of the electric field in the vicinity of the cell membrane taking into account the screening effect of dissolved ions in the cytoplasm was performed. It was discovered that fields crossing the electroporation threshold occurred at a distance of 2 nm or less, supporting the claim that electroporation could be a possible mechanism for fusion pore formation.     

Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K(+) channel.

Three-dimensional computer modeling is used to further investigate the hypothesis forwarded in the accompanying paper of an evolutionary relationship between four related families of K(+) sympoter proteins and the superfamily of K(+) channel proteins. Atomic-scale models are developed for the transmembrane regions of one member from each of the three more distinct symporter families, i.e., a TrkH protein from Escherichia coli, a KtrB protein from Aquifex aeolicus, and a Trk1,2 protein from Schizosaccharomyces pombe. The portions of the four consecutive M1-P-M2 motifs in the symporters that can be aligned with K(+) channel sequences are modeled directly from the recently determined crystal structure of the KcsA K(+) channel from Streptomyces lividans. The remaining portions are developed using our previously accumulated theoretical modeling criteria and principles. Concurrently, the use of these criteria and principles is further supported by the now verified predictions of our previous K(+) channel modeling efforts and the degree to which they are satisfied by the known structure of the KcsA protein. Thus the observed ability of the portions of the symporter models derived from the KcsA crystal structure to also satisfy the theoretical modeling criteria provides additional support for an evolutionary link with K(+) channel proteins. Efforts to further satisfy the criteria and principles suggest that the symporter proteins from fungi and plants (i.e., Trk1,2 and HKT1) form dimeric and/or tetrameric complexes in the membrane. Furthermore, analysis of the atomic-scale models in relation to the sequence conservation within and between the protein families suggests structural details for previously proposed mechanisms for the linked symport of K(+) with Na(+) and H(+). Suggestions are also given for experiments to test these structures and hypotheses.