Role of image force in a water pore of K+ channel in the channel permeability--nonlocal electrostatic effects

A modified Parsegian formula for the calculation of the mage force in a water pore, which takes into account nonlocal electrostatic effects, has been applied to the analysis of changes in the permeability of K+ channels. It has been shown that the channel permeability increases 3 x 10(3) times as the width of the pore increases from 4 to 10 angstroms when the energy is calculated by the modified Parsegian formula and only 16 times if the classical Parsegian formula is used. We conclude that the image force in a water pore within K+ channels plays an important part in the change of channel permeability. Accounting for nonlocal electrostatic effects indicates that the permeability of the K+ channel is more strongly related to the size of the water pore than previously assumed by the original Parsegian formula.     

Molecular docking study of the binding of aminopyridines within the K<Superscript>+</Superscript> channel

We present a molecular docking study aimed to identify the binding site of protonated aminopyridines for the blocking of voltage dependent K⁺ channels. Several active aminopyridines are considered: 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 3,4-diaminopyridine, and 4-aminoquinoleine. We apply the AutoDock force field with a lamarckian genetic algorithm, using atomic charges for the ligands derived from the electrostatic potential obtained at the B3LYP/cc-pVDZ level. We find a zone in the α-subunit of the K⁺ channel bearing common binding sites. This zone corresponds to five amino acids comprised between residuals Thr107 and Ala111, in the KcsA K⁺ channel (1J95 pdb structure). The 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, and 3,4-diaminopyridine bind to the carboxylic oxygens of Thr107 and Ala111. In all cases aminopyridines are perpendicular to the axis of the pore. 4-aminoquinoleine binds to the carboxylic oxygen of Ala111. Due to its large size, the molecular plane is parallel to the axis of the pore. The charge distributions and the structures of the binding complexes suggest that the interaction is driven by formation of several hydrogen bonds. We find 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, and 3,4-diaminopyridine with similar binding energy. Considering the standard error of the estimate of the AutoDock force field, this energy should lie, as a rough estimation, in the interval 3–7 kcal mol⁻¹. On the other hand, 4-aminoquinoleine seems to have a smaller binding energy. Figure Three-dimensional structure of the complex between 4-AQH⁺ and the binding sites of the K⁺ pore. Only the amino acid sequence from Thr107 to Ala111 is considered. Two different representations are included. In the left, the Thr107 position is marked. The right representation shows the CO oxygens of the peptide bond as spacefilled structures.     

Interaction of local anesthetics with the K+ channel pore domain

Local anesthetics and related drugs block ionic currents of Na⁺, K⁺ and Ca²⁺ conducted across the cell membrane by voltage-dependent ion channels. Many of these drugs bind in the permeation pathway, occlude the pore and stop ion movement. However channel-blocking drugs have also been associated with decreased membrane stability of certain tetrameric K⁺ channels, similar to the destabilization of channel function observed at low extracellular K⁺ concentration. Such drug-dependent stability may result from electrostatic repulsion of K⁺ from the selectivity filter by a cationic drug molecule bound in the central cavity of the channel. In this study we used the pore domain of the KcsA K⁺ channel protein to test this hypothesis experimentally with a biochemical assay of tetramer stability and theoretically by computational simulation of local anesthetic docking to the central cavity. We find that two common local anesthetics, lidocaine and tetracaine, promote thermal dissociation of the KcsA tetramer in a K⁺-dependent fashion. Docking simulations of these drugs with open, open-inactivated and closed crystal structures of KcsA yield many energetically favorable drug-channel complexes characterized by nonbonded attraction to pore-lining residues and electrostatic repulsion of K⁺. The results suggest that binding of cationic drugs to the inner cavity can reduce tetramer stability of K⁺ channels.     

K<Superscript>+</Superscript> Channels of Squid Giant Axons Open by an Osmotic Stress in Hypertonic Solutions Containing Nonelectrolytes

In hypertonic solutions made by adding nonelectrolytes, K⁺ channels of squid giant axons opened at usual asymmetrical K⁺ concentrations in two different time courses; an initial instantaneous activation (I _IN) and a sigmoidal activation typical of a delayed rectifier K⁺ channel (I _D). The current–voltage relation curve for I _IN was fitted well with Goldman equation described with a periaxonal K⁺ concentration at the membrane potential above −10 mV. Using the activation–voltage curve obtained from tail currents, K⁺ channels for I _IN are confirmed to activate at the membrane potential that is lower by 50 mV than those for I _D. Both I _IN and I _D closed similarly at the holding potential below −100 mV. The logarithm of I _IN/I _D was linearly related with the osmolarity for various nonelectrolytes. Solute inaccessible volumes obtained from the slope increased with the nonelectrolyte size from 15 to 85 water molecules. K⁺ channels representing I _D were blocked by open channel blocker tetra-butyl ammonium (TBA) more efficiently than in the absence of I _IN, which was explained by the mechanism that K⁺ channels for I _D were first converted to those for I _IN by the osmotic pressure and then blocked. So K⁺ channels for I _IN were suggested to be derived from the delayed rectifier K⁺ channels. Therefore, the osmotic pressure is suggested to exert delayed-rectifier K⁺ channels to open in shrinking rather hydrophilic flexible parts outside the pore than the pore itself, which is compatible with the recent structure of open K⁺ channel pore.     

Modulation of Rabbit Corneal Epithelial Cell Proliferation by Growth Factor-regulated K<Superscript>+</Superscript> Channel Activity

We characterized the dependence of the mitogenic response by rabbit corneal epithelial (RCE) cells to serum containing growth factors on K⁺ channel activation. Using both cell-attached and nystatin-perforated patch-clamp configurations, a K⁺ channel was identified whose current-voltage relationship is linear with a single-channel conductance of 31 pS. Its activity was barely detectable following 24 h serum starvation. Exposure of starved cells to either 10% FBS, 5 ng/ml epidermal growth factor (EGF) or 2 nM endothelin-1 (ET-1) continuously increased its activity within 30 min by 40%, 54% and 29%, respectively. EGF and ET-1 in combination had additive effects on such activity. Application of 100 µM 4-aminopyridine (4-AP), a K⁺ channel blocker, inhibited serum-stimulated K⁺ channel activity by 85%. DNA synthesis was markedly stimulated by serum, whereas incubation with either 4-AP (200 µM) or Ba²⁺ (1 mM) suppressed this increase by 51% and 23%, respectively, whereas 5 mM tetra ethyl ammonium (TEA) had no effect. Taken together, growth factor-induced increases in proliferation are dependent on K⁺ channel stimulation. As the increases in K⁺ channel activity induced by ET-1 and EGF were additive, these mitogens may stimulate K⁺ channel activity through different signaling pathways linked to their cognate receptors.     

Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

ATP-regulated K+ channel in mitochondria: Pharmacology and function

Mitochondria from several tissues contain a potassium-specific channel similar to the ATP-regulated K⁺ (K_ATP) channel of the plasma membrane. The mitochondrial channel shares with the plasma membrane K_ATP channel the sensitivity to sulfonylurea derivatives and some other blockers as well as to channel openers of diverse chemical character. In contrast to the plasma membrane channel, which is blocked by free ATP, the mitochondrial K_ATP channel reconstituted into liposomes requires the ATP-Mg complex for inhibition. The mitochondrial K_ATP channel, possibly in a concerted action with other K⁺ permeability pathways, plays an important role in mitochondrial volume control. Its function in the regulation of the components of the protonmotive force is also suggested.     

Energy distribution and ion selectivity of the bacterial potassium channel

The ion selectivity of the bacterial potassium channel K_CSA is explained upon comparing the energy characteristics of the interaction of cations (Li⁺, Na⁺, K⁺) with atoms of the selectivity filter of the protein pore. Quantum-chemical calculations reveal a deeper potential well for potassium ions, which accounts for preferred K⁺ permeation. It is shown that the conventional methods with AMBER, CHARMM, OPLS force fields in standard parametrization as well as partial re-parametrization give incorrect estimates of ion energy distribution in the channel.     

Structure of the pore-helix of the hERG K<Superscript>+</Superscript> channel

The hERG K⁺ channel undergoes rapid inactivation that is mediated by ‘collapse’ of the selectivity filter, thereby preventing ion conduction. Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology with channels with known crystal structures. In the present work, we determined structural features of a peptide from the pore loop region of hERG (residues 600–642) in both sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC) micelles using NMR spectroscopy. A complete structure calculation was done for the peptide in DPC, and the localization of residues inside the micelles were analysed by using a water-soluble paramagnetic reagent with both DPC and SDS micelles. The pore-helix in the hERG peptide was only two–four residues longer at the N-terminus, compared with the pore helices seen in the crystal structures of other K⁺ channels, rather than the seven residues suggested from previous NMR studies. The helix in the peptide spanned the same residues in both micellar environments despite a difference in the localization inside the respective micelles. To determine if the extension of the length of the helix was affected by the hydrophobic environment in the two types of micelles, we compared NMR and X-ray crystallography results from a homologous peptide from the voltage gated potassium channel, KcsA.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

Molecular dynamics study of the KcsA channel at 2.0-Å resolution: stability and concerted motions within the pore

The stability of the KcsA channel accommodating more than one ion in the pore has been studied with molecular dynamics. We have used the very last X-ray structure of the KcsA channel at 2.0-Å resolution determined by Zhou et al. [Nature 414 (2001) 43]. In this channel, six of the seven experimentally evidenced sites have been considered. We show that the protein remains very stable in the presence of four K⁺ ions (three in the selectivity filter and one in the cavity). The locations and the respective distances of the different K⁺ ions and water molecules (W), calculated within our KWKWKK sequence, also fits well with the experimental observations. The analysis of the K⁺ ions and water molecules displacements shows concerted file motions on the simulated time scale (≈1 ns), which could act as precursor to the diffusion of K⁺ ions inside the channel. A simple one-dimensional dynamical model is used to interpret the concerted motions of the ions and water molecules in the pore leading ultimately to ion transfer.     

Identification and characterization of a novel bacterial ATP-sensitive K<Superscript>+</Superscript> channel

Five bacterial species that are most likely to have putative prokaryotic inward rectifier K⁺ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K⁺ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K⁺ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K⁺ current with characteristics similar to those of the ATP-sensitive K⁺ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.     

Acute hypoxia differentially regulates K<Superscript>+</Superscript> channels. Implications with respect to cardiac arrhythmia

The first ion channels demonstrated to be sensitive to changes in oxygen tension were K⁺ channels in glomus cells of the carotid body. Since then a number of hypoxia-sensitive ion channels have been identified. However, not all K⁺ channels respond to hypoxia alike. This has raised some debate about how cells detect changes in oxygen tension. Because ion channels respond rapidly to hypoxia it has been proposed that the channel is itself an oxygen sensor. However, channel function can also be modified by thiol reducing and oxidizing agents, implicating reactive oxygen species as signals in hypoxic events. Cardiac ion channels can also be modified by hypoxia and redox agents. The rapid and slow components of the delayed rectifier K⁺ channel are differentially regulated by hypoxia and -adrenergic receptor stimulation. Mutations in the genes that encode the subunits for the channel are associated with Long QT syndrome and sudden cardiac death. The implications with respect to effects of hypoxia on the channel and triggering of cardiac arrhythmia will be discussed.     

Differential expression of KCNQ1 K+ channel in tubular cells of frog kidney

The aim of this study was to evaluate KCNQ1 K⁺ channel expression in the frog kidney of Rana esculenta. KCNQ1 K⁺ channel, also known as KvLQT1, is the pore forming α-subunit of the I_Ks K⁺ channel, a delayed rectifier voltage-gated K⁺ channel, which has an important role in water and salt transport in the kidney and gastrointestinal tract. The expression of KCNQ1 K⁺ channel along tubular epithelium differs from species to species. In the present study the expression of KCNQ1 K⁺ channel in the frog kidney has been demonstrated by immunohistochemistry. The presence of KCNQ1 K⁺ channel was demonstrated in the epithelial cells of distal convoluted tubule and collecting duct. However, the pattern of expression of KCNQ1 K⁺ channel differs between distal convoluted tubules and collecting duct. All epithelial cells of distal convoluted tubules revealed basolateral expression of KCNQ1 K⁺ channel. On the contrary, only the single cells of collecting duct, probably intercalated cells, showed diffuse cell surface staining with antibodies against KCNQ1 K⁺ channel. These findings suggest that KCNQ1 K⁺ channel has cell-specific roles in renal potassium ion transport.Key words: KCNQ1 K⁺ channel, rana esculenta, frog kidney, immunohistochemistry.     

Tectonics of a K+ channel: The importance of the N-terminus for channel gating

The small K⁺ channel Kcv represents the pore module of complex potassium channels. It was found that its gating can be modified by sensor domains, which are N-terminally coupled to the pore. This implies that the short N-terminus of the channel can transmit conformational changes from upstream sensors to the channel gates. To understand the functional role of the N-terminus in the context of the entire channel protein, we apply combinatorial screening of the mechanical coupling and long-range interactions in the Kcv potassium channel by reduced molecular models. The dynamics and mechanical connections in the channel complex show that the N-terminus is indeed mechanically connected to the pore domain. This includes a long rang coupling to the pore and the inner and outer transmembrane domains. Since the latter domains host the two gates of the channel, the data support the hypothesis that mechanical perturbation of the N-terminus can be transmitted to the channel gates. This effect is solely determined by the topology of the channel; sequence details only have an implicit effect on the coarse-grained dynamics via the fold and not through biochemical details at a smaller scale. This observation has important implications for engineering of synthetic channels on the basis of a K⁺ channel pore.     

Ion permeation in a K+ channel inChara australis: Direct evidence for diffusion limitation of ion flow in a maxi-K channel

Summary We report a study of a potassium-selective channel in the membrane delineating cytoplasmic drops fromChara australis. The relatively large conductance (170 pS in 150 mol/m³ (mm) KCl), high ion selectivity (P _Cl/P _K=0.015±0.01) and voltagedependent kinetics of this channel indicate that it is a type of maxi-K channel commonly found in animal cells but not previously detected in any plant cell.The current-voltage (I/V) characteristic of these channels was examined in drop-attached and in excised outside-out patches using the patch-clamp technique, over the unusually large voltage range of –250 to 200 mV. TheI/V characteristic is nonlinear and shows saturation at extreme voltages; the current also saturates at high [K⁺]. In solutions with symmetrical KCl concentrations the saturation behavior of the current is asymmetrical. The permeability of the channel depends on whether it is observed in excised or in drop-attached membrane patches.Here we investigate the main factors affecting the permeation of K⁺ ions through this maxi-K channel. We present the first direct evidence for the importance of diffusion external to the pore in limiting ion flow through maxi-K channels. The data are consistent with an ion translocation mechanism whose current is limited (i) at high voltages by ion diffusion external to the pore and (ii) at high [K⁺] by the maximum transport rate of the channel. We fit the data to a diffusion-limited pore model in which the pore exhibits saturation described by Michaelis-Menten kinetics with aK _m=50±25 mol/m³ andG _max=300±20 pS.     

Hydration valve controlled non-selective conduction of Na and K in the NaK channel

The Na⁺ and K⁺ channels are essential to neural signaling, but our current knowledge at the atomic level is mainly limited to the conducting mechanism of K⁺. Unlike a K⁺ channel having four equivalent K⁺-binding sites in its selectivity filter, a NaK channel has a vestibule in the middle part of its selectivity filter, and can conduct both Na⁺ and K⁺ ions. However, the underlying mechanism for non-selective ion conduction in NaK remains elusive. Here we find four small grottos connecting with the vestibule of the NaK selectivity filter, which form a vestibule-grotto complex perpendicular to the filter pore with a few water molecules within it. It is shown that two or more of the water molecules coming to the vestibule to coordinate the cation are necessary for conducting both Na⁺ and K⁺ ions, while only one water molecule in the vestibule will obstruct ion permeation. Thus, the complex with the aid of interior water movement forms a dynamic hydration valve which is flexible in conveying different cations through the vestibule. Similar exquisite hydration valve mechanisms are expected to be utilized by other non-selective cation channels, and the results should shed new light on the importance of water in neural signaling.     

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.     

The k/na selectivity of a cation channel in the plasma membrane of root cells does not differ in salt-tolerant and salt-sensitive wheat species

The characteristics of cation outward rectifier channels were studied in protoplasts from wheat root (Triticum aestivum L. and Triticum turgidum L.) cells using the patch clamp technique. The cation outward rectifier channels were voltage-dependent with a single channel conductance of 32 ± 1 picosiemens in 100 millimolar KCl. Whole-cell currents were dominated by the activity of the cation outward rectifiers. The time- and voltage-dependence of these currents was accounted for by the summed behavior of individual channels recorded from outside-out detached patches. The K⁺/Na⁺ permeability ratio of these channels was measured in a salt-sensitive and salt-tolerant genotype of wheat that differ in rates of Na⁺ accumulation, using a voltage ramp protocol on protoplasts in the whole-cell configuration. Permeability ratios were calculated from shifts in reversal potentials following ion substitutions. There were no significant differences in the K⁺/Na⁺ permeability ratios of these channels in root cells from either of the two genotypes tested. The permeability ratio for K⁺/Cl⁻ was greater than 50:1. The K⁺/Na⁺ permeability ratio averaged 30:1, which is two to four times more selective than the same type of channel in guard cells and suspension culture cells. Lowering the Ca²⁺ concentration in the bath solution to 0.1 millimolar in the presence of 100 millimolar Na⁺ had no significant effect on the K⁺/Na⁺ permeability ratios of the channel. It seems unlikely that the mechanism of salt tolerance in wheat is based on differences in the K⁺/Na⁺ selectivity of these channels.     

Crucial points within the pore as determinants of K⁺ channel conductance and gating

While selective for K⁺, K⁺ channels vary significantly among their rate of ion permeation. Here, we probe the effect of steric hindrance and electrostatics within the ion conduction pathway on K⁺ permeation in the MthK K⁺ channel using structure-based mutagenesis combined with single-channel electrophysiology and X-ray crystallography. We demonstrate that changes in side-chain size and polarity at Ala88, which forms the constriction point of the open MthK pore, have profound effects on single-channel conductance as well as open probability. We also reveal that the negatively charged Glu92s at the intracellular entrance of the open pore form an electrostatic trap, which stabilizes a hydrated K⁺ and facilitates ion permeation. This electrostatic attraction is also responsible for intracellular divalent blockage, which renders the channel inward rectified in the presence of Ca²⁺. In light of the high structural conservation of the selectivity filter, the size and chemical environment differences within the portion of the ion conduction pathway other than the filter are likely the determinants for the conductance variations among K⁺ channels.