Ion Selectivity of the KcsA Channel: A Perspective from Multi-Ion Free Energy Landscapes

Potassium (K⁺) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K⁺ through cell membranes. Comprising five specific cation binding sites (S₀-S₄) formed by the backbone carbonyl groups of conserved residues common to all K⁺ channels, the narrow selectivity filter allows fast conduction of K⁺ while being highly selective for K⁺ over Na⁺. To extend our knowledge of the microscopic mechanism underlying selectivity in K⁺ channels, we characterize the free energy landscapes governing the entry and translocation of a Na⁺ or a K⁺ from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K⁺ in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K⁺ or Na⁺ in binding site S₀ or S₁ are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na⁺ than for K⁺, implying that the kinetics of ion conduction is slower when a Na⁺ enters the pore. Third, the region corresponding to binding site S₂ near the center of the narrow pore emerges as the most selective for K⁺ over Na⁺. In particular, while there is a stable minimum for K⁺ in site S₂, Na⁺ faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S₂ in the selectivity filter of K⁺ channels.     

Quantum calculations on water in the KcsA channel cavity with permeant and non-permeant ions

Different ions in the pore of the KcsA channel behave differently, and we relate this to their solvation. We show that the selectivity is dependent, in part, on the solvation in the cavity (sometimes referred to as the vestibule, it is the region containing water molecules between the intracellular gate and the selectivity filter at the extracellular end of the pore). We have shown earlier that potassium is more dependent at the upper end of the cavity region on solvation by the threonines there, while sodium ion has more water molecules as ligands. In addition, sodium ion is placed asymmetrically, while potassium is nearly exactly symmetric with respect to the four-fold symmetry of the channel. We have now extended these calculations to rubidium and cesium ions, and find that rubidium solvation resembles that of potassium (and both are permeant ions), while cesium resembles sodium (and both are non-permeant), in terms of the geometry of up to eight hydrating, and four non-hydrating, water molecules. In each case, a maximum of 12 water molecules are relevant to the calculation. The placement of the water molecules in the two cases is essentially the same as found from the electron density in the X-ray structure of Zhou and MacKinnon. For Na⁺ and K⁺, we show that energy decreases from bulk to the cavity to the lowest position in the selectivity filter (accurate energy could not be calculated for the heavier ions). A separate calculation shows that fixing the Na⁺ ion at the position of the K⁺ minimum, followed by re-optimization produced a significantly modified system, not something that could be produced by thermal fluctuations. Moving the K⁺ into the Na⁺ position in the upper cavity led to a small increase in energy, ≈ 3 k_BT, but was accompanied by large shifts in the positions of hydrating waters, which would create a major kinetic barrier. Therefore, thermal fluctuations could not invalidate the conclusions of the main calculations.     

Kir4.1 K+ channels are regulated by external cations

The inwardly rectifying potassium channel (Kir), Kir4.1 mediates spatial K⁺-buffering in the CNS. In this process the channel is potentially exposed to a large range of extracellular K⁺ concentrations ([K⁺]_o). We found that Kir4.1 is regulated by K⁺_o. Increased [K⁺]_o leads to a slow (mins) increase in the whole-cell currents of Xenopus oocytes expressing Kir4.1. Conversely, removing K⁺ from the bath solution results in a slow decrease of the currents. This regulation is not coupled to the pH_i-sensitive gate of the channel, nor does it require the presence of K67, a residue necessary for K⁺_o-dependent regulation of Kir1.1. The voltage-dependent blockers Cs⁺ and Ba²⁺ substitute for K⁺ and prevent deactivation of the channel in the absence of K⁺_o. Cs⁺ blocks and regulates the channel with similar affinity, consistent with the regulatory sites being in the selectivity-filter of the channel. Although both Rb⁺ and NH₄⁺ permeate Kir4.1, only Rb⁺ is able to regulate the channel. We conclude that Kir4.1 is regulated by ions interacting with specific sites in the selectivity filter. Using a kinetic model of the permeation process we show the plausibility of the channel’s sensing the extracellular ionic environment through changes in the selectivity occupancy pattern, and that it is feasible for an ion with the selectivity properties of NH₄⁺ to permeate the channel without inducing these changes.     

Ion–protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy

An understanding of ion–protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion–protein interactions in the KcsA selectivity filter. A band at 1680 cm^?1 in the amide I region would be a marker band for the ion occupancy of K⁺, Rb⁺, and Cs⁺ in the filter. The band at 1627 cm^?1 observed in both Na⁺ and Li⁺ conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K⁺ ions provides quantitative information on the ion affinity of the selectivity filter.     

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

ConclusionThe equilibrium ion-binding properties of ion channels and transporters can be difficult to discern from crystal structures alone, as proteins often adopt different lowest energy states depending on the ions bound. In cases where transport is slow, their inherent ion-binding preferences can be used to infer their transport preferences. However, in cases where transport is fast, the transport selectivity can hide their equilibrium preferences by accentuating the kinetics of ions hopping through a channel over its inherent ion-binding preferences. Thus, depending on the arrangement of ion-binding sites in a channel’s selectivity filter, one can achieve either selective or nonselective ion transport.The equilibrium K⁺ selectivity of some nonselective channels suggests a potential mechanism whereby they could evolve into a fast K⁺-selective channel. K⁺ channels and nonselective channels like CNG and HCN are related to one another in both sequence and structure, suggesting an evolutionary link between them. Swap experiments show that only a few mutations separate a nonselective channel from a K⁺-selective channel. One might imagine an evolutionary path between these channels in which the equilibrium preference for a K⁺ ion in a nonselective channel evolves into a K⁺-selective channel through these few mutations to create the selective ion queue. Alternatively, a slow single-ion channel with an equilibrium and transport preference for K⁺ ions could be transformed into a fast multi-ion channel through mutations that create a queue of K⁺-selective ion-binding sites, as is seen in most K⁺ channels studied to date.In the case of multi-ion selectivity filters, such as those found in K⁺ channels, the selectivity filter can be viewed as the active site that interacts with different queues of ions and water molecules. At least three properties emerge from multi-ion queues: (1) high conductance by reducing the affinity of multiple bound ions versus single ions; (2) high selectivity by allowing disfavored ions time to dissociate back into solution; and, consequently, (3) robust selectivity in an environment where ion concentrations can change. For transporters and carriers, the equilibrium preference and slow transport naturally create robust selectivity. In all these cases, equilibrium-based ion selectivity is achieved by slowing transport enough so that the disfavored ion is able to dissociate back into solution before transport takes place.     

Effects of Permeant Cations on K+ Channel Gating in Nerve Axons Revisited

An increase in extracellular potassium ion concentration, K _o , significantly slows the potassium channel deactivation rate in squid giant axons, as previously shown. Surprisingly, the effect does not occur in all preparations which, coupled with the voltage independence of this result in preparations in which it does occur, suggests that it is mediated at a site outside of the electric field of the channel, and that this site is accessible to potassium ions in some preparations, but not in others. In other words, the effect does not appear to be related to occupancy of the channel by potassium ions. This conclusion is supported by a four-barrier, three-binding site model of single file diffusion through the channel in which one site, at most, is unoccupied by a potassium ion (single-vacancy model). The model is consistent with current-voltage relations with various levels of K _o , and, by definition, with multiple occupancy by K⁺. The model predicts that occupancy of any given site is essentially independent of K _o (or K _i ). The effects of extracellular Rb⁺ and Cs⁺ on gating are strongly voltage dependent, and they were observed in all preparations investigated. Consequently, the mechanism underlying these results would appear to be different from that which underlies the effect of K⁺ on gating. In particular, the effect of Rb⁺ on gating is reduced by strong hyperpolarization, which in the context of the occupancy hypothesis, is consistent with the voltage dependence of the current-voltage relation in the presence of Rb⁺. The primary, novel, finding in this study is that the effects of Cs⁺ are counterintuitive in this regard. Specifically, the slowing of channel deactivation rate by Cs⁺ is also reduced by hyperpolarization, similar to the Rb⁺ results, whereas blockade is enhanced, which is seemingly inconsistent with the concept that occupancy of the channel by Cs⁺ underlies the effect of this ion on gating. This result is further elucidated by barrier modeling of the current-voltage relation in the presence of Cs⁺. Received: 19 December 1995/Revised: 10 June 1996     

Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

Potassium and sodium ions in a potassium channel studied by molecular dynamics simulations

We have performed simulations of both a single potassium ion and a single sodium ion within the pore of the bacterial potassium channel KcsA. For both ions there is a dehydration energy barrier at the cytoplasmic mouth suggesting that the crystal structure is a closed conformation of the channel. There is a potential energy barrier for a sodium ion in the selectivity filter that is not seen for potassium. Radial distribution functions for both ions with the carbonyl oxygens of the selectivity filter indicate that sodium may interact more tightly with the filter than does potassium. This suggests that the key to the ion selectivity of KcsA is the greater dehydration energy of Na⁺ ions, and helps to explain the block of KcsA by internal Na⁺ ions.     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na⁺ and K⁺ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na⁺ and K⁺. These structural rearrangements facilitate entry of K⁺ ions into the selectivity filter and permeation through the channel, and rejection of Na⁺ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K⁺. Estimates of the K⁺/Na⁺ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na⁺ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.     

Iberiotoxin and clofilium regulate hyperactivation,acrosome reaction,and ion homeostasis synergistically during human sperm capacitation

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K⁺ currents in human sperm (I_KSper) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca²⁺, K⁺, Cl⁻, and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K⁺]_i, [Cl⁻]_i, and pH_i, but a decrease in [Ca²⁺]_i. Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K⁺]_i, [Cl⁻]_i, and pH_i, and the decrease in [Ca²⁺]_i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.     

Chemically induced K+ conduction noise in squid axon

Internal perfusion of tetraethylammonium ions (TEA) in squid axons produces a significant high frequency noise component. Although internal TEA suppresses the potassium conductance (G _K) noise at relatively low frequencies, it induces high frequency noise which exceeds the intensity of the normal potassium and sodium noise. In addition, the induced noise is dependent on the presence of internal potassium ions (K⁺) suggesting that this source of noise arises from a modulation of the K⁺ conductance due to the blocking and unblocking of the K⁺ channel. The simplest model describing the TEA data is a two-step sequential pseudo-unimolecular reaction where TEA binds during an open conductance state. A unit channel conductance of 2 pS is estimated from the TEA data as well as noise induced by triethyldecylammonium (TEDA) ions. Thus, these data are consistent with the hypothesis that the channel is blocked whenever the quaternary ammonium ion binding site, located near or within the K⁺ channel, is occupied.     

Ca2+-Activated K+ channel from human erythrocyte membranes: Single channel recification and selectivity

Summary The Ca²⁺-activated K⁺ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K⁺, Rb⁺, and NH ₄ ⁺ , respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K⁺ (23.5 pS±3.2)>NH ₄ ⁺ (14.2 pS±1.2)>Rb⁺ (11.4 pS±1.8). With low extracellular K⁺ concentrations (substitution with Na⁺) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K⁺ and Na⁺ enhanced rectification were observed due to a Na⁺ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (P _K/P _X) was calculated: K⁺ (1.0)>Rb⁺ (1.4±0.1)>NH ₄ ⁺ (8.5±1.3)>Li⁺(>50); Na⁺ (>110); Cs⁺ (5). Li⁺, Na⁺, and Cs⁺ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl⁺ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process.     

Ion conductance and ion selectivity of potassium channels in snail neurones

Summary Delayed potassium channels were studied in internally perfused neurone somata from land snails. Relaxation and fluctuation analysis of this class of ion channels revealed Hodgkin-Huxley type K channels with an average single channel conductance ( _K) of 2.40±0.15 pS. The conductance of open channels is independent of voltage and virtually all K channels seem to be open at maximum K conductance (g _K) of the membrane. Voltage dependent time constants of activation ofg _K, calculated from K current relaxation and from cut-off frequencies of power spectra, are very similar indicating dominant first-order kinetics. Ion selectivity of K channels was studied by ion substitution in the external medium and exhibited the following sequence: T1⁺>K⁺>Rb⁺>Cs⁺>NH ₄ ⁺ >Li⁺>Na⁺. The sequence of the alkali cations does not conform to any of the sequences predicted by Eisenman's theory. However, the data are well accommodated by a new theory assuming a single rate-limiting barrier that governs ion movement through the channel.This paper is dedicated to the memory of Walther Wilbrandt.     

The Impermeant Ion Methylammonium Blocks K+ and NH+ 4 Currents through KAT1 Channel Differently: Evidence for Ion Interaction in Channel Permeation

The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different ions in the external medium. With either K⁺, NH⁺ ₄ or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K⁺ and, to a lesser extent, to NH⁺ ₄ but not to the slightly bigger, methylated analogue of NH⁺ ₄, MA. Substituting NH⁺ ₄ for K⁺ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that channel operation depends on the transported substrate. In mixed solution (50 mm K⁺, 50 mm MA) MA inhibited K⁺ current in a voltage-independent manner. The maximum block did not exceed 50% of the K⁺ current. In contrast, when NH⁺ ₄ was the permeant ion (50 mm NH⁺ ₄, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages. The latter block could be partially overcome by the addition of K⁺ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different affinities for binding sites on their permeation pathway. Received: 6 October 1997/Revised: 28 January 1998     

Computer simulations of neuron-glia interactions mediated by ion flux

Extracellular potassium concentration, [K⁺]_o, and intracellular calcium, [Ca²⁺]_i, rise during neuron excitation, seizures and spreading depression. Astrocytes probably restrain the rise of K⁺ in a way that is only partly understood. To examine the effect of glial K⁺ uptake, we used a model neuron equipped with Na⁺, K⁺, Ca²⁺ and Cl⁻ conductances, ion pumps and ion exchangers, surrounded by interstitial space and glia. The glial membrane was either “passive”, incorporating only leak channels and an ion exchange pump, or it had rectifying K⁺ channels. We computed ion fluxes, concentration changes and osmotic volume changes. Increase of [K⁺]_o stimulated the glial uptake by the glial 3Na/2K ion pump. The [K⁺]_o flux through glial leak and rectifier channels was outward as long as the driving potential was outwardly directed, but it turned inward when rising [K⁺]_o/[K⁺]_i ratio reversed the driving potential. Adjustments of glial membrane parameters influenced the neuronal firing patterns, the length of paroxysmal afterdischarge and the ignition point of spreading depression. We conclude that voltage gated K⁺ currents can boost the effectiveness of the glial “potassium buffer” and that this buffer function is important even at moderate or low levels of excitation, but especially so in pathological states.     

Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana

Plant roots accumulate K⁺ over a range of external concentrations. Root cells have evolved at least two parallel plasma-membrane K⁺ transporters which operate at millimolar and micromolar external [K⁺]: high-affinity K⁺ uptake is energised by symport with H⁺, while low-affinity uptake is assumed to occur via ion channels. To determine the role of ion channels in low-affinity K⁺ uptake, a characterisation of the principal K⁺-selective ion channels in the plasma membrane of Arabidopsis thaliana (L.) Heynh. cv. Columbia roots was undertaken. Two classes of K⁺-selective channels were frequently observed: one inward (IRC) and one outward (ORC) rectifying with unitary conductances of 5 pS, 20 pS (IRCs) and 15 pS (ORC), measured in symmetrical 10 mM KCl. The dominant IRC (5 pS) and ORC (15 pS) were highly cation-selective (P_Cl P_K < 0.025) but less selective amongst monovalent cations (P_NaP_K0.17–0.3). Both the IRC and the ORC were blocked by Ba²⁺, Cs⁺ and tetra-ethyl-ammonium, whereas 4-aminopyridine and quinidine selectively inhibited the ORC. The ORC open probability was steeply voltage-dependent and ORC activation potentials were close to the potassium equilibrium potential (E_K+), enabling ORCs to conduct mainly outward, but occasionally inward, K⁺ current. By contrast, gating of the 5-pS IRC was weakly voltageependent and IRC gating was invariably restricted to membrane potentials more negative than E_K+, ensuring K⁺ transport was always inwardly directed. Studies on channel activity were conducted for a large number of root cells grown at two levels of external [K⁺], one where K⁺ uptake is likely to be principally through channels (6 mM K⁺) and one where it must be energised (100 M K⁺). Shifting growth conditions from high to low K⁺ did not affect single-channel properties such as conductance and selectivity, nor the manifestation of the ORC and 20-pS IRC, but led to enhanced activity of the 5-pS IRC. The enhanced activity of the 5-pS IRC was mirrored by a parallel increase in unidirectional ⁸⁶Rb⁺ influx after low-K⁺ growth, clearly indicating a dominant role of this particular channel in K⁺ uptake at supra millimolar external [K⁺].Abbreviations E_K+ potassium equilibrium potential - E_m membrane potential - HK high [K⁺] - IRC inward rectifying channel - LK low [K⁺] - ORC outward rectifying channel - TEA tetra-ethyl-ammonium Financial support was provided by the Biotechnology and Biological Sciences Research Council (Grant PG87/529) and by the European Union (Framework III, Biotechnology Programme).     

Conformational Dynamics in the Selectivity Filter of KcsA in Response to Potassium Ion Concentration

Conformational change in the selectivity filter of KcsA as a function of ambient potassium concentration is studied with solid-state NMR. This highly conserved region of the protein is known to chelate potassium ions selectively. We report solid-state NMR chemical shift fingerprints of two distinct conformations of the selectivity filter; significant changes are observed in the chemical shifts of key residues in the filter as the potassium ion concentration is changed from 50 mM to 1 μM. Potassium ion titration studies reveal that the site-specific K_d for K⁺ binding at the key pore residue Val76 is on the order of ∼ 7 μM and that a relatively high sample hydration is necessary to observe the low-K⁺ conformer. Simultaneous detection of both conformers at low ambient potassium concentration suggests that the high-K⁺ and low-K⁺ states are in slow exchange on the NMR timescale (k_ex < 500 s^− 1). The slow rate and tight binding for evacuating both inner sites simultaneously differ from prior observations in detergent in solution, but agree well with measurements by electrophysiology and appear to result from our use of a hydrated bilayer environment. These observations strongly support a common assumption that the low-K⁺ state is not involved in ion transmission, and that during transmission one of the two inner sites is always occupied. On the other hand, these kinetic and thermodynamic characteristics of the evacuation of the inner sites certainly could be compatible with participation in a control mechanism at low ion concentration such as C-type inactivation, a process that is coupled to activation and involves closing of the outer mouth of the channel.     

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.     

Nonselective Conduction in a Mutated NaK Channel with Three Cation-Binding Sites

The NaK channel is a cation-selective protein with similar permeability for K⁺ and Na⁺ ions. Crystallographic structures are available for the wild-type and mutated NaK channels with different numbers of cation-binding sites. We have performed a comparison between the potentials of mean force governing the translocation of K⁺ ions and mixtures of one Na⁺ and three K⁺ ions in a mutated NaK channel with only three cation-binding sites (NaK-CNG). Since NaK-CNG is not selective for K⁺ over Na⁺, analysis of its multi-ion potential energy surfaces can provide clues about how selectivity originates. Comparison of the potentials of mean force of NaK-CNG and K⁺-selective channels yields observations that strongly suggest that the number of contiguous ion binding sites in a single-file mechanism is the key determinant of the channel’s selectivity properties, as already proposed by experimental studies. We conclude that the presence of four binding sites in K⁺-selective channels is essential for highly selective and efficient permeation of K⁺ ions, and that a key difference between K⁺-selective and nonselective channels is the absence/presence of a binding site for Na⁺ ions at the boundary between S2 and S3 in the context of multi-ion permeation events.