Ion binding affinity in the cavity of the KcsA potassium channel

The hydrophobic cell membrane interior presents a large energy barrier for ions to permeate. Potassium channels reduce this barrier by creating a water-filled cavity at the middle of their ion conduction pore to allow ion hydration and by directing the C-terminal "end charge" of four alpha-helices toward the water-filled cavity. Here we have studied the interaction of monovalent cations with the cavity of the KcsA K(+) channel using X-ray crystallography. In these studies, Tl(+) was used as an analogue for K(+) and the total ion-stabilization energy for Tl(+) in the cavity was estimated by measuring its binding affinity. Binding affinity for the Na(+) ion was also measured, revealing a weak selectivity ( approximately 7-fold) favoring Tl(+) over Na(+). The structures of the cavity containing Na(+), K(+), Tl(+), Rb(+), and Cs(+) are compared. These results are consistent with a fairly large (more negative than -100 mV) electrostatic potential inside the cavity, and they also imply the presence of a weak nonelectrostatic component to a cation's interaction with the cavity.     

The interfacial lipid binding site on the potassium channel KcsA is specific for anionic phospholipids

Lipid binding to the potassium channel KcsA from Streptomyces lividans has been studied using quenching of the fluorescence of Trp residues by brominated phospholipids. It is shown that binding of phospholipids to nonannular lipid binding sites on KcsA, located one each at the four protein-protein interfaces in the tetrameric structure, is specific for anionic phospholipids, zwitterionic phosphatidylcholine being unable to bind at the sites. The binding constant for phosphatidylglycerol of 3.0 ± 0.7 mol fraction⁻¹ means that in a membrane containing ~20 mol% phosphatidylglycerol, as in the Escherichia coli inner membrane, the nonannular sites will be ~37% occupied by phosphatidylglycerol. The binding constant for phosphatidic acid is similar to that for phosphatidylglycerol but binding constants for phosphatidylserine and cardiolipin are about double those for phosphatidylglycerol. Binding to annular sites around the circumference of the KcsA tetramer are different on the extracellular and intracellular faces of the membrane. On the extracellular face of the membrane the binding constants for anionic lipids are similar to those for phosphatidylcholine, the lack of specificity being consistent with the lack of any marked clusters of charged residues on KcsA close to the membrane on the extracellular side. In contrast, binding to annular sites on the intracellular side of the membrane shows a distinct structural specificity, with binding of phosphatidic acid and phosphatidylglycerol being stronger than binding of phosphatidylcholine, whereas binding constants for phosphatidylserine and cardiolipin are similar to that for phosphatidylcholine. It is suggested that this pattern of binding follows from the pattern of charge distribution on KcsA on the intracellular side of the membrane.     

Multiple binding sites for fatty acids on the potassium channel KcsA

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 μM.     

Phosphatidic acid plays a special role in stabilizing and folding of the tetrameric potassium channel KcsA

In this study, we investigated how the presence of anionic lipids influenced the stability and folding properties of the potassium channel KcsA. By using a combination of gel electrophoresis, tryptophan fluorescence and acrylamide quenching experiments, we found that the presence of the anionic lipid phosphatidylglycerol (PG) in a phosphatidylcholine (PC) bilayer slightly stabilized the tetramer and protected it from trifluoroethanol-induced dissociation. Surprisingly, the presence of phosphatidic acid (PA) had a much larger effect on the stability of KcsA and this lipid, in addition, significantly influenced the folding properties of the protein. The data indicate that PA creates some specificity over PG, and that it most likely stabilizes the tetramer via both electrostatic and hydrogen bond interactions.     

A computational study of ion binding and protonation states in the KcsA potassium channel

We report results from microscopic molecular dynamics and free energy perturbation simulations of the KcsA potassium channel based on its experimental atomic structure. Conformational properties of selected amino acid residues as well as equilibrium positions of K(+) ions inside the selectivity filter and the internal water cavity are examined. Positions three and four (counting from the extracellular site) in the experimental structure correspond to distinctly separate binding sites for K(+) ions inside the selectivity filter. The protonation states of Glu71 and Asp80, which are close to each other and to the selectivity filter, as well as K(+) binding energies are determined using free energy perturbation calculations. The Glu71 residue which is buried inside a protein cavity is found to be most stable in the neutral form while the solvent exposed Asp80 is ionized. The channel altogether exothermically binds up to three ions, where two of them are located inside the selectivity filter and one in the internal water cavity. Ion permeation mechanisms are discussed in relation to these results.     

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites.Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.     

Modeling permeation energetics in the KcsA potassium channel

The thermodynamics of cation permeation through the KcsA K(+) channel selectivity filter is studied from the perspective of a physically transparent semimicroscopic model using Monte Carlo free energy integration. The computational approach chosen permits dissection of the separate contributions to ionic stabilization arising from different parts of the channel (selectivity filter carbonyls, single-file water, cavity water, reaction field of bulk water, inner helices, ionizable residues). All features play important roles; their relative significance varies with the ion's position in the filter. The cavity appears to act as an electrostatic buffer, shielding filter ions from structural changes in the inner pore. The model exhibits K(+) vs. Na(+) selectivity, and roughly isoenergetic profiles for K(+) and Rb(+), and discriminates against Cs(+), all in agreement with experimental data. It also indicates that Ba(2+) and Na(+) compete effectively with permeant ions at a site near the boundary between the filter and the cavity, in the vicinity of the barium blocker site.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

Molecular dynamics study of the KcsA potassium channel

The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately (1)/(3) of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel.     

Interactions of phospholipids with the potassium channel KcsA

The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.     

Rapid intracellular TEA block of the KcsA potassium channel

Intracellular tetraethylammonium (TEA) inhibition was studied at the single-channel level in the KcsA potassium channel reconstituted in planar lipid bilayers. TEA acts as a fast blocker (resulting in decreased current amplitude) with an affinity in the 75 mM range even at high bandwidth. Studies over a wide voltage range reveal that TEA block has a complex voltage-dependence that also depends on the ionic conditions. These observations are examined in the context of permeation models to extend our understanding of the coupling between permeant ions and TEA blockade.     

Mechanisms of tetraethylammonium ion block in the KcsA potassium channel

We report results from automated docking and microscopic molecular dynamics simulations of the tetraethylammonium (TEA) complexes with KcsA. Binding modes and energies for TEA binding at the external and internal sides of the channel pore are examined utilising the linear interaction energy method. Effects of the channel ion occupancy (based on our previous results for the ion permeation mechanisms) on the binding energies are considered. Calculations show that TEA forms stable complexes at both the external and internal entrances of the selectivity filter. Furthermore, the effects of the Y82V mutation are evaluated and the results show, in agreement with experimental data, that the mutant has a significantly reduced binding affinity for TEA at the external binding site, which is attributed to stabilising hydrophobic interactions between the ligand and the tyrosines.     

Inactivation of the KcsA potassium channel explored with heterotetramers

The tetrameric prokaryotic potassium channel KcsA is activated by protons acting on the intracellular aspect of the protein and inactivated through conformational changes in the selectivity filter. Inactivation is modulated by a network of interactions within each protomer between the pore helix and residues at the external entrance of the channel. Inactivation is suppressed by the E71A mutation, which perturbs the stability of this network. Here, cell-free protein synthesis followed by protein purification by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to produce heterotetramers of KcsA that contain different combinations of wild-type and E71A subunits. Single-channel recordings from these heterotetramers reveal how the network of interactions in individual protomers affects ionic conduction and channel inactivation, suggesting that the latter is a cooperative process.     

Intermolecular functional coupling between phosphoinositides and the potassium channel KcsA

Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K⁺ channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol. However, there has been no quantitative analysis of the correlation between direct lipid binding and the functional modification of KcsA, and it is unknown whether PIPns modulate KcsA function. Here, using contact bubble bilayer recording, we observed that the open probability of KcsA increased significantly (from about 10% to 90%) when the membrane inner leaflet contained only a small percentage of PIPns. In addition, we found an increase in the electrophysiological activity of KcsA correlated with a larger number of negative charges on PIPns. We further analyzed the affinity of the direct interaction between PIPns and KcsA using microscale thermophoresis and observed a strong correlation between direct lipid binding and the functional modification of KcsA. In conclusion, our approach was able to reconstruct the direct modification of KcsA by PIPns, and we propose that it can also be applied to elucidate the mechanism of modification of other ion channels by PIPns.     

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA

All K⁺-channels are stabilized by K⁺-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K⁺-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K⁺-channel.     

Tetraethylammonium binding to the outer mouth of the KcsA potassium channel: implications for ion permeation

Extracellular tetraethylammonium (TEA+) inhibits the current carried out by K+ ions in potassium channels. Structural models of wild-type (WT) and Y82C KcsA K+ channel/TEA+ complexes are here built using docking procedures, electrostatics calculations and molecular dynamics simulations. The calculations are based on the structure determined by Doyle et al. (11) Our calculations suggest that in WT, the TEA+ cation turns binds at the outer mouth of the selectivity filter, stabilized by electrostatic and hydrophobic interactions with the four Tyr82 side chains. Replacement of Tyr82 with Cys causes a decrease of the affinity of the cation for the channel, consistently with the available site-directed mutagenesis data (16). An MD simulation in which K+ replaces TEA+ provides evidence that TEA+ binding site can accommodate a potassium ion, in agreement with the high-resolution structure recently reported by Zhou et al. (20)     

Water and potassium dynamics inside the KcsA K(+) channel

Molecular dynamics simulations and electrostatic modeling are used to investigate structural and dynamical properties of the potassium ions and of water molecules inside the KcsA channel immersed in a membrane-mimetic environment. Two potassium ions, initially located in the selectivity filter binding sites, maintain their position during 2 ns of dynamics. A third potassium ion is very mobile in the water-filled cavity. The protein appears engineered so as to polarize water molecules inside the channel cavity. The resulting water induced dipole and the positively charged potassium ion within the cavity are the key ingredients for stabilizing the two K(+) ions in the binding sites. These two ions experience single file movements upon removal of the potassium in the cavity, confirming the role of the latter in ion transport through the channel.     

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with approximately 180 SDS molecules per channel. K(+) ions bind to the micelle-solubilized channel with a K(D) of 3 +/- 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.     

Interactions of anionic phospholipids and phosphatidylethanolamine with the potassium channel KcsA

Fluorescence quenching methods have been used to study interactions of anionic phospholipids with the potassium channel KcsA from Streptomyces lividans. Quenching of the Trp fluorescence of KcsA reconstituted into mixtures of dioleoylphosphatidylcholine (DOPC) and an anionic phospholipid with dibromostearoyl chains is more marked at low mole fractions of the brominated anionic phospholipid than is quenching in mixtures of dibromostearoylphosphatidylcholine and nonbrominated anionic lipid. The quenching data are consistent with two classes of binding site for lipid on KcsA, one set corresponding to annular binding sites around KcsA to which DOPC and two-chain anionic phospholipids bind with similar affinities, the other set (non-annular sites) corresponding to sites at which anionic phospholipids can bind but from which DOPC is either excluded or binds with very low affinity. The binding constant for tetraoleoylcardiolipin at the annular sites is significantly less than that for DOPC, being comparable to that for dioleoylphosphatidylethanolamine. Tetraoleoylcardiolipin binds with highest affinity to the non-annular sites, the affinity for dioleoylphosphatidylglycerol being the lowest. The affinity for dioleoylphosphatidylserine decreases at high ionic strength, suggesting that electrostatic interactions between the anionic phospholipid headgroup and positively charged residues on KcsA are important for binding at the non-annular site. The effect of ionic strength on the binding of phosphatidic acid is less marked than on phosphatidylserine. The value of the binding constant for the non-annular site depends on the extent of Trp fluorescence quenching following from binding at the non-annular site. It is suggested that the non-annular site to which binding is detected in the fluorescence quenching experiments corresponds to the binding site for phosphatidylglycerol detected at monomer-monomer interfaces in x-ray diffraction studies.