The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11)?M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH?5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.     

Influence of lipids on membrane assembly and stability of the potassium channel KcsA

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two-dimensional 1H-nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long alpha-helix containing eight turns along nearly the full length of the peptide except for four N-terminal residues and six C-terminal residues. The electrostatic surface map shows that the N-terminal segment of the alpha-helix, residues 5-22, is an amphipathic alpha-helix with a clear separation of hydrophobic and hydrophilic faces, and that the C-terminal segment of the alpha-helix, residues 23-36, is a hydrophobic alpha-helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N-terminal segment of the alpha-helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.     

Anionic phospholipid interactions with the potassium channel KcsA: simulation studies

Molecular dynamics (MD) simulations have been used to unmask details of specific interactions of anionic phospholipids with intersubunit binding sites on the surface of the bacterial potassium channel KcsA. Crystallographic data on a diacyl glycerol fragment at this site were used to model phosphatidylethanolamine (PE), or phosphatidylglycerol (PG), or phosphatidic acid (PA) at the intersubunit binding sites. Each of these models of a KcsA-lipid complex was embedded in phosphatidyl choline bilayer and explored in a 20 ns MD simulation. H-bond analysis revealed that in terms of lipid-protein interactions PA > PG > PE and revealed how anionic lipids (PG and PA) bind to a site provided by two key arginine residues (R(64) and R(89)) at the interface between adjacent subunits. A 27 ns simulation was performed in which KcsA (without any lipids initially modeled at the R(64)/R(89) sites) was embedded in a PE/PG bilayer. There was a progressive specific increase over the course of the simulation in the number of H-bonds of PG with KcsA. Furthermore, two specific PG binding events at R(64)/R(89) sites were observed. The phosphate oxygen atoms of bound PG formed H-bonds to the guanidinium group of R(89), whereas the terminal glycerol H-bonded to R(64). Overall, this study suggests that simulations can help identify and characterize sites for specific lipid interactions on a membrane protein surface.     

Characterization of the C-terminal domain of a potassium channel from Streptomyces lividans (KcsA)

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.     

Effects of conducting and blocking ions on the structure and stability of the potassium channel KcsA

This article reports on the interaction of conducting (K(+)) and blocking (Na(+)) monovalent metal ions with detergent-solubilized and lipid-reconstituted forms of the K(+) channel KcsA. Monitoring of the protein intrinsic fluorescence reveals that the two ions bind competitively to KcsA with distinct affinities (dissociation constants for the KcsA.K(+) and KcsA.Na(+) complexes of approximately 8 and 190 mm, respectively) and induce different conformations of the ion-bound protein. The differences in binding affinity as well as the higher K(+) concentration bathing the intracellular mouth of the channel, through which the cations gain access to the protein binding sites, should favor that only KcsA.K(+) complexes are formed under physiological-like conditions. Nevertheless, despite such prediction, it was also found that concentrations of Na(+) well below its dissociation constant and even in the presence of higher K(+) concentrations, cause a remarkable decrease in the protein thermal stability and facilitate thermal dissociation into subunits of the tetrameric KcsA, as concluded from the temperature dependence of the protein infrared spectra and from gel electrophoresis, respectively. These latter observations cannot be explained based on the occupancy of the binding sites from above and suggest that there must be additional ion binding sites, whose occupancy could not be detected by fluorescence and in which the affinity for Na(+) must be higher or at least similar to that of K(+). Moreover, cation binding as reported by means of fluorescence does not suffice to explain the large differences in free energy of stabilization involved in the formation of the KcsA.Na(+) and KcsA.K(+) complexes, which for the most part should arise from synergistic effects of the ion-mediated intersubunit interactions.     

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.     

Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1

In this study, we compared the channel intrinsic stability of the bacterial K⁺-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at ～45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.     

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.     

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.     

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.     

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.     

The influence of a membrane environment on the structure and stability of a prokaryotic potassium channel, KcsA

The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1-125 KcsA reconstituted into native-like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent-protein crystal conditions.     

Intracellular domains interactions and gated motions of IKS potassium channel subunits

Voltage‐gated K⁺ channels co‐assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore‐forming subunits with KCNE1 β subunits generates the repolarizing K⁺ current I_KS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life‐threatening long or short QT syndromes. Here, we studied the interactions and voltage‐dependent motions of I_KS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage‐clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C‐terminus interacts with the coiled‐coil helix C of the Kv7.1 tetramerization domain. This association is important for I_KS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant‐negative C‐terminal domain. On channel opening, the C‐termini of Kv7.1 and KCNE1 come close together. Co‐expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K⁺ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C‐termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.     

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.     

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.     

hERG potassium channel gating is mediated by N- and C-terminal region interactions

Human ether-á-go-go-related gene (hERG) potassium channels have voltage-dependent closing (deactivation) kinetics that are unusually slow. A Per-Arnt-Sim (PAS) domain in the cytoplasmic N-terminal region of hERG regulates slow deactivation by making a direct interaction with another part of the hERG channel. The mechanism for slow deactivation is unclear, however, because the other regions of the channel that participate in regulation of deactivation are not known. To identify other functional determinants of slow deactivation, we generated hERG channels with deletions of the cytoplasmic C-terminal regions. We report that hERG channels with deletions of the cyclic nucleotide-binding domain (CNBD) had accelerated deactivation kinetics that were similar to those seen in hERG channels lacking the PAS domain. Channels with dual deletions of the PAS domain and the CNBD did not show further acceleration in deactivation, indicating that the PAS domain and the CNBD regulate deactivation by a convergent mechanism. A recombinant PAS domain that we previously showed could directly regulate PAS domain-deleted channels did not regulate channels with dual deletions of the PAS domain and CNBD, suggesting that the PAS domain did not interact with CNBD-deleted channels. Biochemical protein interaction assays showed that glutathione S-transferase (GST)-PAS (but not GST) bound to a CNBD-containing fusion protein. Coexpression of PAS domain-deleted subunits (with intact C-terminal regions) and CNBD-deleted subunits (with intact N-terminal regions) resulted in channels with partially restored slow deactivation kinetics, suggesting regulatory intersubunit interactions between PAS domains and CNBDs. Together, these data suggest that the mechanism for regulation of slow deactivation in hERG channels is an interaction between the N-terminal PAS domain and the C-terminal CNBD.     

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.     

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.