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.     

Monte Carlo simulations of ionic channel selectivity

Monte Carlo simulations have been developed to study the selectivityof ionic channels in biological membranes. The channel is consideredto be in either of two possible states: (i) densely packed withions, the ions moving in single file in one direction, or alternatively,(ii) sparsely packed, where holes could appear at any particulartime thereby allowing bidirectional movement of ions. The twomodels enable us to envisage a quantitative flux of permeableions in the presence of smaller sized ions, taking into considerationtheir concentrations in the bulk solutions, the ion-channelinteractions and probability with which they fill up the channel.The programs are written in FORTRAN-77 (MS-FORTRAN) for an IBM-compatiblepersonal computer. From the simulation results we observe anenzymatic function of the channel and also note that the smallersized ions tend to block the movement of permeable ions. Thesimulations represent a technique for visualization of the factorsthat decide ionic permeability and help in manipulating theireffects with ease and speed which would otherwise involve intricateexperimental setups. Received on September 7, 1990; accepted on January 14, 1991     

Mechanism of K+/Na+ selectivity in potassium channels from the perspective of the non-selective bacterial channel NaK

Studies of the non-selective NaK channel as a model system provided detailed structural and functional insights into different factors that modulate selective ion conductance. The combination of structural and functional studies of WT- and mutant forms of NaK channel show that apparent conservation of the multiple binding sites in the canonical selectivity filter of K-channel is essential component of the observed robust Na (+) discrimination.     

The predominant role of coordination number in potassium channel selectivity

Potassium channels are exquisitely selective, allowing K+ to pass across cell membranes while blocking other ion types. Here we demonstrate that the number of carbonyl oxygen atoms that surround permeating ions is the most important factor in determining ion selectivity rather than the size of the pore or the strength of the coordinating dipoles. Although the electrostatic properties of the coordinating ligands can lead to Na+ or K+ selectivity at some values of the dipole moment, no significant selectivity arises at the specific value of the dipole moment for carbonyl groups found in potassium channels when the ligands have complete freedom. Rather, we show that the main contribution to selectivity arises from slight constraints on the conformational freedom of the channel protein that limit the number of carbonyl oxygen atoms to a value better suited to K+ than Na+, despite the pore being flexible. This mechanism provides an example of a general framework for explaining ion discrimination in a range of natural and synthetic macromolecules in which selectivity is controlled by the number of coordinating ligands in addition to their dipole moment.     

The cation selectivity filter of the bacterial sodium channel,NaChBac

The Bacillus halodurans voltage-gated sodium-selective channel (NaChBac) (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001b. SCIENCE: 294:2372-2375), is an ideal candidate for high resolution structural studies because it can be expressed in mammalian cells and its functional properties studied in detail. It has the added advantage of being a single six transmembrane (6TM) orthologue of a single repeat of mammalian voltage-gated Ca(2+) (Ca(V)) and Na(+) (Na(V)) channels. Here we report that six amino acids in the pore domain (LESWAS) participate in the selectivity filter. Replacing the amino acid residues adjacent to glutamatic acid (E) by a negatively charged aspartate (D; LEDWAS) converted the Na(+)-selective NaChBac to a Ca(2+)- and Na(+)-permeant channel. When additional aspartates were incorporated (LDDWAD), the mutant channel resulted in a highly expressing voltage-gated Ca(2+)-selective conductance.     

The ligand-sensitive gate of a potassium channel lies close to the selectivity filter

Potassium channels selectively conduct K⁺ ions across cell membranes and have key roles in cell excitability. Their opening and closing can be spontaneous or controlled by membrane voltage or ligand binding. We used Ba²⁺ as a probe to determine the location of the ligand-sensitive gate in an inwardly rectifying K⁺ channel (Kir6.2). To a K⁺ channel, Ba²⁺ and K⁺ are of similar sizes, but Ba²⁺ blocks the pore by binding within the selectivity filter. We found that internal Ba²⁺ could still access its binding site when the channel was shut, which indicates that the ligand-sensitive gate lies above the Ba²⁺-block site, and thus within or above the selectivity filter. This is in marked contrast to the voltage-dependent gate of K_V channels, which is located at the intracellular mouth of the pore.     

Ion selectivity mechanism in a bacterial pentameric ligand-gated ion channel

The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ∼11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2′) at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl⁻ in the middle of the pore for both GLIC and the E-2′A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.     

Effects of some potassium channel blockers on the ionic currents in myelinated nerve

The effects of some potassium channel blockers on the ionic currents and on the so-called K(+)-depolarization in intact myelinated nerve fibres were studied. 4-AP, and in particular, Flaxedil, proved to be selective K(+)-current blockers. However, TEA, a crown ether (DCH18C6), a longchained triethylammonium compound (C10-TriEA), capsaicin, and the extract from the medicinal herb Ruta graveolens proved not to be selective K(+)-current blockers; they all block Na(+)-currents as well, although to a lesser extent. The sodium inactivation curve did not change under TEA and Flaxedil but was shifted on the potential axis in negative direction by DCH18C6, 4-AP, capsaicin and the Ruta extract whereas C10-TriEA caused a shift of both sodium inactivation and activation parameters in positive direction. Regarding to the kinetics of the persisting K(+)-current fraction, two different kinds of blockade were found: 1. Unchanged K(+)-kinetic which is typical for the effects of TEA, 4-AP, Flaxedil, and C10-TriEA. 2. Clearly changed K(+)-kinetic, characterized by K(+)-transients; which is typical for the effects of capsaicin and in particular, for those of DCH18C6 and of the Ruta extract. The possibly different modes of action of both groups of blockers are discussed in terms of current models for the action of potassium channel blockers.     

Modulation of C-type inactivation by K+ at the potassium channel selectivity filter.

With prolonged or repetitive activation, voltage-gated K+ channels undergo a slow (C-type) inactivation mechanism, which decreases current flow through the channel. Previous observations suggest that C-type inactivation results from a localized constriction in the outer mouth of the channel pore and that the rate of inactivation is controlled by the-rate at which K+ leaves an unidentified binding site in the pore. We have functionally identified two K+ binding sites in the conduction pathway of a chimeric K+ channel that conducts Na+ in the absence of K+. One site has a high affinity for K+ and contributes to the selectivity filter mechanism for K+ over Na+. Another site, external to the high-affinity site, has a lower affinity for K+ and is not involved in channel selectivity. Binding of K+ to the high-affinity binding site slowed inactivation. Binding of cations to the external low-affinity site did not slow inactivation directly but could slow it indirectly, apparently by trapping K+ at the high-affinity site. These data support a model whereby C-type inactivation involves a constriction at the selectivity filter, and the constriction cannot proceed when the selectivity filter is occupied by K+.     

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.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3-1000 microM) induced a smooth inward current that was competitively inhibited by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 microM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (Er) of -6.1 +/- 0.5 mV. In cerebellar mRNA-injected oocytes; Er was nearly identical (-5.1 +/- 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd++ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3–1000 µ.M) induced a smooth inward current that was competitively inhibted by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 µM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (E_r) of -6.1 ± 0.5 mV In cerebellar mRNA-injected oocytes; E_r was nearly identical (-5.1 ± 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd⁺⁺ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.     

Role of hydrophobic and ionic forces in the movement of S4 of the Shaker potassium channel

Abstract

Voltage-gated ion (K⁺, Na⁺, Ca²⁺) channels contain a pore domain (PD) surrounded by four voltage sensing domains (VSD). Each VSD is made up of four transmembrane helices, S1–S4. S4 contains 6–7 positively charged residues (arginine/lysine) separated two hydrophobic residues, whereas S1–S3 contribute to two negatively charged clusters. These structures are conserved among all members of the voltage-gated ion channel family and play essential roles in voltage gating. The role of S4 charged residues in voltage gating is well established: During depolarization, they move out of the membrane electric field, exerting a mechanical force on channel gates, causing them to open. However, the role of the intervening hydrophobic residues in voltage sensing is unclear. Here we studied the role of these residues in the prototypical Shaker potassium channel. We have altered the physicochemical properties of both charged and hydrophobic positions of S4 and examined the effect of these modifications on the gating properties of the channel. For this, we have introduced cysteines at each of these positions, expressed the mutants in Xenopus oocytes, and examined the effect of in situ addition of charge, via Cd²⁺, on channel gating by two-electrode voltage clamp. Our results reveal a face of the S4 helix (comprising residues L358, L361, R365 and R368) where introduction of charge at hydrophobic positions destabilises the closed state and removal of charges from charged positions has an opposite effect. We propose that hydrophobic residues play a crucial role in limiting gating to a physiological voltage range.     

Kinetic approach with ab initio MO method on ionic selectivity and size in sodium channel

Three kinds of models for ionic selectivity and size of the filter in sodium channel have been treated by using ab initio molecular orbital (MO) calculations with MINI-3 and MIDI-3* basis sets. A three-components system, HCO2M-H2O (M = Li+, Na+ or K+), is acceptable for describing experimental facts well. Thermochemical parameters obtained from harmonic vibrational analysis with MINI-3 basis sets, for the translocation of the permeant metal cations in the HCO2M-H2O system, are that the activation enthalpies for Li+, Na+ and K+ are 7.0, 6.4 and 23.4 kJ/mol, and also the free energies of activation are 10.6, 1.5 and 19.0 kJ/mol, respectively. These results are qualitatively in good correspondence with experimental facts of the ion selectivity of the channel. One of water molecule was found to have a key role in the translocation of the permeant cations.     

Altered ion channel conductance and ionic selectivity induced by large imposed membrane potential pulse.

The effects of large magnitude transmembrane potential pulses on voltage-gated Na and K channel behavior in frog skeletal muscle membrane were studied using a modified double vaseline-gap voltage clamp. The effects of electroconformational damage to ionic channels were separated from damage to lipid bilayer (electroporation). A 4 ms transmembrane potential pulse of -600 mV resulted in a reduction of both Na and K channel conductivities. The supraphysiologic pulses also reduced ionic selectivity of the K channels against Na+ ions, resulting in a depolarization of the membrane resting potential. However, TTX and TEA binding effects were unaltered. The kinetics of spontaneous reversal of the electroconformational damage of channel proteins was found to be dependent on the magnitude of imposed membrane potential pulse. These results suggest that muscle and nerve dysfunction after electrical shock may be in part caused by electroconformational damage to voltage-gated ion channels.     

Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel

KcsA is a proton-activated K⁺ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K⁺, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K⁺ selectivity, and permitted Na⁺ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.