Ion selectivity of the Kat1 K+ channel pore

Kat1 is a highly selective inward-rectifying K⁺ channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K⁺ channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K⁺ over Na⁺. Mutants of this class frequently displayed sensitivity to the competing ion Cs⁺. This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K⁺/Na⁺ selectivity, but selectivity over Cs⁺, and possibly a wide spectrum of potential competing ions.     

A mammalian two pore domain mechano-gated S-like K+ channel.

Aplysia S-type K+ channels of sensory neurons play a dominant role in presynaptic facilitation and behavioural sensitization. They are closed by serotonin via cAMP-dependent phosphorylation, whereas they are opened by arachidonic acid, volatile general anaesthetics and mechanical stimulation. We have identified a cloned mammalian two P domain K+ channel sharing the properties of the S channel. In addition, the recombinant channel is opened by lipid bilayer amphipathic crenators, while it is closed by cup-formers. The cytoplasmic C-terminus contains a charged region critical for chemical and mechanical activation, as well as a phosphorylation site required for cAMP inhibition.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

A two-state homology model of the hERG K+ channel: application to ligand binding

Homology models based on available K+ channel structures have been used to construct a multiple state representation of the hERG cardiac K+ channel. These states are used to capture the flexibility of the channel. We show that this flexibility is essential in order to correctly model the binding affinity of a set of diverse ligands. Using this multiple state approach, a binding affinity model was constructed for set of known hERG channel binders. The predicted pIC50s are in good agreement with experiment (RMSD: 0.56 kcal/mol). In addition, these calculations provide structures for the bound ligands that are consistent with published mutation studies. These computed ligand bound complex structures can be used to guide synthesis of analogs with reduced hERG liability.     

Modeling P-loops domain of sodium channel: homology with potassium channels and interaction with ligands

A large body of experimental data on Na+ channels is available, but the interpretation of these data in structural terms is difficult in the absence of a high-resolution structure. Essentially different electrophysiological and pharmacological properties of Na+ and K+ channels and poor identity of their sequences obstruct homology modeling of Na+ channels. In this work, we built the P-loops model of the Na+ channel, in which the pore helices are arranged exactly as in the MthK bacterial K+ channel. The conformation of the selectivity-filter region, which includes residues in positions -2 through +4 from the DEKA locus, was shaped around rigid molecules of saxitoxin and tetrodotoxin that are known to form multiple contacts with this region. Intensive Monte Carlo minimization that started from the MthK-like conformation produced practically identical saxitoxin- and tetrodotoxin-based models. The latter was tested to explain a wide range of experimental data that were not used at the model building stage. The docking of tetrodotoxin analogs unambiguously predicted their optimal orientation and the interaction energy that correlates with the experimental activity. The docking of mu-conotoxin produced a binding model consistent with experimentally known toxin-channel contacts. Monte Carlo-minimized energy profiles of tetramethylammonium pulled through the selectivity-filter region explain the paradoxical experimental data that this organic cation permeates via the DEAA but not the AAAA mutant of the DEKA locus. The model is also consistent with earlier proposed concepts on the Na+ channel selectivity as well as Ca2+ selectivity of the EEEE mutant of the DEKA locus. Thus, the model integrates available experimental data on the Na+ channel P-loops domain, and suggests that it is more similar to K+ channels than was believed before.     

Modeling by homology of RNA binding domain in A1 hnRNP protein

Eukaryotic nuclear RNA binding proteins share a common sequence motif thought to be implicated in RNA binding. One of the two domains present in A1 hnRNP protein, has been modelled by homology in order to make a prediction of the main features of the RNA binding site. Acylphosphatase (EC 3.6.1.7) was selected as template for the modeling experiment. The predicted RNA binding site is a beta-sheet containing the two RNP consensus sequences as well as lysines and arginines conserved among the family.     

Folding of the voltage-gated K+ channel T1 recognition domain

Voltage-gated K(+) channels (Kv) are tetramers whose assembly is coordinated in part by a conserved T1 recognition domain. Although T1 achieves its quaternary structure in the ER, nothing is known about its acquisition of tertiary structure. We developed a new folding assay that relies on intramolecular cross-linking of pairs of cysteines engineered at the folded T1 monomer interface. Using this assay, we show directly that the T1 domain is largely folded while the Kv protein is still attached to membrane-bound ribosomes. The ER membrane facilitates both folding and oligomerization of Kv proteins. We show that folding and oligomerization assays can be used to study coupling between these two biogenic events and diagnose defects in assembly of Kv channels.     

The pore domain outer helix contributes to both activation and inactivation of the HERG K+ channel

Ion flow in many voltage-gated K(+) channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K(+) channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe(551) to Trp(563), affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala(561), His(562), Ala(565), Trp(568), and Ile(571)), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

We investigated the effects of changing extracellular K⁺ concentrations on block of the weak inward-rectifier K⁺ channel Kir1.1b (ROMK2) by the three intracellular cations Mg²⁺, Na⁺, and TEA⁺. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K⁺ in the inside (cytoplasmic) solution and 11 mM K⁺ in the outside (extracellular) solution, these three cations blocked K⁺ currents with a range of apparent affinities (K_i (0) = 1.6 mM for Mg²⁺, 160 mM for Na⁺, and 1.8 mM for TEA⁺) but with similar voltage dependence (zδ = 0.58 for Mg²⁺, 0.71 for Na⁺, and 0.61 for TEA⁺) despite having different valences. When external K⁺ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg²⁺ but not for Na⁺ or TEA⁺. In contrast, the N152Y mutation increased the affinity for block by TEA⁺ but not for Na⁺ or Mg²⁺. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg²⁺ but had a small effect on that by Na⁺. TEA⁺ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K⁺ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K⁺ are explained by shifts in the occupancy of sites within the selectivity filter by K⁺ ions.     

A model for identifying HERG K+ channel blockers

Acquired long QT syndrome (LQTS) occurs frequently as a side effect of blockade of cardiac HERG K(+) channels by commonly used medications. A large number of structurally diverse compounds have been shown to inhibit K(+) current through HERG. There is considerable interest in developing in silico tools to filter out potential HERG blockers early in the drug discovery process. We describe a binary classification model that combines a 2D topological similarity filter with a 3D pharmacophore ensemble procedure to discriminate between HERG actives and inactives with an overall accuracy of 82%, with false negative and false positive rates of 29% and 15%, respectively. This model should be generally applicable in virtual library counterscreening against HERG.     

K+ binding sites and interactions between permeating K+ ions at the external pore mouth of an inward rectifier K+ channel (Kir2.1).

The arginine at position 148 is highly conserved in the inward rectifier K+ channel family. Increases of external pH decrease the single-channel conductance in mutant R148H of the Kir2.1 channel (arginine is mutated into histidine) but not in the wild type channel. Moreover, in 100 mM external K+, varying external pH induced biphasic changes of open channel noise, which peaks at around pH 7.4 in the R148H mutant but not in the wild type channel. The maximum single-channel conductances are higher in the wild type channel and R148H mutant at pH 6.0 than those in the R148H mutant at pH 7.4. However, the maximal conductance is achieved with much lower external [K+] for the latter. Interestingly, the single-channel conductances and open channel noise of the wild type channel at pH 6. 0 and the R148H mutant at pH 6.0 and 7.4 become the same in [K+] = 10 mM. These results indicate that the residue at position 148 is accessible to the external H+ and probably is involved in the formation of two K+ binding sites in the external pore mouth. Effective repulsion between permeating K+ ions in this area requires a positive charge at position 148, and such K+-K+ interaction is the essential mechanism underlying high K+ conduction rate through the Kir2.1 channel pore.     

K+ channels lacking the 'tetramerization' domain: implications for pore structure

The difficulty of obtaining high-resolution structures of integral membrane proteins has been a frustrating barrier to understanding the membrane-based functions of living cells. The mere handful of such structures stands out in dismal contrast to the cornucopia of water-soluble proteins comprehensible at the atomic level. Nevertheless, crystallographically tractable preparations of aqueous domains of membrane proteins have provided molecular insight into phenomena as varied as chemotaxis, immune cell responses to antigens, viral infectivity and cellular synthesis of ATP. Recently, the first structural glimpse of a neuronal ion channel was reported - the T1, or 'tetramerization,' domain of a Shaker-type voltage-gated K+ channel at 1.6 A resolution. The isolated domain associates into a water-soluble four-fold symmetric homotetramer. This structure prompted the novel, provocative proposal that the T1 domain is an essential component of the ion permeation pathway, forming a previously unsuspected ion-coordinating constriction on the cytoplasmic side of the channel and acting as the receptor for the pore-blocking 'ball and chain' inactivation peptide. It has also been commonly conjectured that the T1 domain is required for tetramerization in the channel maturation process. By studying the detailed properties of Shaker K+ channels in which the T1 domain is deleted, we show all these proposals to be invalid. The structure of the T1 domain expressed in isolation is therefore unlikely to mirror in detail its structure when attached to the ion-conducting channel.     

Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.     

Atomic constraints between the voltage sensor and the pore domain in a voltage-gated K+ channel of known structure

In voltage-gated K(+) channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn(2+) or Cd(2+) with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by approximately 7-8 A and rotated counterclockwise by 37 degrees along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.     

A homology model of the pore domain of a voltage-gated calcium channel is consistent with available SCAM data

In the absence of x-ray structures of calcium channels, their homology models are used to rationalize experimental data and design new experiments. The modeling relies on sequence alignments between calcium and potassium channels. Zhen et al. (2005. J. Gen. Physiol. doi:10.1085/jgp.200509292) used the substituted cysteine accessibility method (SCAM) to identify pore-lining residues in the Ca_v2.1 channel and concluded that their data are inconsistent with the symmetric architecture of the pore domain and published sequence alignments between calcium and potassium channels. Here, we have built K_v1.2-based models of the Ca_v2.1 channel with 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET)-modified engineered cysteines and used Monte Carlo energy minimizations to predict their energetically optimal orientations. We found that depending on the position of an engineered cysteine in S6 and S5 helices, the ammonium group in the long flexible MTSET-modified side chain can orient into the inner pore, an interface between domains (repeats), or an interface between S5 and S6 helices. Different local environments of equivalent positions in the four repeats can lead to different SCAM results. The reported current inhibition by MTSET generally decreases with the predicted distances between the ammonium nitrogen and the pore axis. A possible explanation for outliers of this correlation is suggested. Our calculations rationalize the SCAM data, validate one of several published sequence alignments between calcium and potassium channels, and suggest similar spatial dispositions of S5 and S6 helices in voltage-gated potassium and calcium channels.     

Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+.

The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Analysis of the wild-type current now shows that: (i) [K+]o suppresses a very slowly relaxing component, accelerating activation; (ii) [K+]o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating. We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the [K+]o sensitivity. Substitution of these residues gave channels with reduced sensitivity to [K+]o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance. In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low [K+]o. The results indicate that these residues are involved in [K+]o sensing. Their situation close to the permeation pathway points to an interaction between gating and permeation.     

Resonance assignment of the cyclic nucleotide binding domain from a cyclic nucleotide-gated K+ channel in complex with cAMP

In order to determine the structure of the 15 kDa cyclic nucleotide binding domain of a cyclic nucleotide-activated K⁺ channel from Mesorhizobium loti and its interaction with cAMP, nearly complete ¹H, ¹³C, and ¹⁵N chemical shifts were assigned.     

Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel.

Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX. If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27.     

Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K+ channel

A series of n-alkyl-bis-alpha,omega-trimethylammonium (bisQn) compounds was synthesized, and their ability to block K+ currents through a K+ channel from sarcoplasmic reticulum was studied. K+ channels were inserted into planar phospholipid membranes, and single-channel K+ currents were measured in the presence of the blocking cations. These bisQn compounds block K+ currents only from the side of the membrane opposite to the addition of SR vesicles (the trans side). The block is dependent on transmembrane voltage, and the effective valence of the block (a measure of this voltage dependence) varies with the methylene chain length. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 1.1 to 0.65; it then remains constant at approximately 0.65 for bisQ5 to bisQ8; the effective valence abruptly increases to 1.2-1.3 for chains of nine carbons and longer. For the compounds of nine carbons and longer, the discrete nature of the block can be observed directly as 'flickering noise" on the open channel. The kinetics of the block were studied for these long-chain blockers. Both blocking and unblocking rates of the blockers vary with chain length, with the blocking rate showing the strongest variation--an increase of 2.8-fold per added methylene group. All of the voltage dependence of the binding equilibrium resides in the blocking rate, and none in the unblocking rate. The results imply that 65% of the voltage drop within the channel occurs over a distance of 6-7A, and that the short-chain blockers bind in a bent-over conformation with both charges deeply inside the channel.