Contribution of ion binding affinity to ion selectivity and permeation in KcsA, a model potassium channel

Ion permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K(+) to this mutant reveals a single set of low-affinity K(+) binding sites, similar to that seen in the binding of K(+) to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K(+) permeation in E71A. Nonetheless, the E71A mutant retains K(+) selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K(+) and also conducts Na(+). Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.     

Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure

Mutation E71A in the bacterial K⁺-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and ~2.5-fold lower affinity for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol (PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel conductance and increases the channel open time and open probability. Taken together, our data suggest that the “flipped” conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding, whereas higher PG content decreases channel conductivity and stabilizes the channel open “flipped” state via electrostatic interaction in the proximity of the channel pore.     

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl⁺) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H⁺). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C_18:1 to C_22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H⁺ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H⁺ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.     

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl⁺) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it''s noninactivating (E71A) KcsA mutant, by extravesicular protons (H⁺). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C_18:1 to C_22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H⁺ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H⁺ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.     

Diverse gating in K channels: Differential role of the pore-helix glutamate in stabilizing the channel pore

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K⁺ (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71–Asp80 carboxyl–carboxylate interaction ‘behind’ the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu–Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K⁺ channels.     

Influence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA

KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Delta125-160 and Delta120-160, as well as 1-125 KcsA obtained from chymotrypsin cleavage of full-length 1-160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120-124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.     

Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction

The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region. We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.     

Functional influence of the pore helix glutamate in the KcsA K+ channel

The E71 residue is buried near the selectivity filter in the KcsA K+ channel and forms a carboxyl-carboxylate bridge with D80. We have investigated the importance of E71 by examining neutralization mutants at this position using biochemical and electrophysiological methods. E71 mutations differentially destabilize the detergent-solubilized tetramer; among them, the E71V neutralization mutant has a relatively subtle effect. The E71V channel displays electrical activity when reconstituted into planar lipid bilayers. In single channel recordings, the mutant retains K+/Na+ selectivity, and its conductance in the outward direction is unaltered. Some conduction properties are changed: inward conductance is increased. Our results show that that the E71 side chain is not a primary determinant of ion selectivity or conduction in the wild-type channel, either directly or through the E71:D80 carboxyl-carboxylate bridge.     

Molecular determinants of gating at the potassium-channel selectivity filter

We show that in the potassium channel KcsA, proton-dependent activation is followed by an inactivation process similar to C-type inactivation, and this process is suppressed by an E71A mutation in the pore helix. EPR spectroscopy demonstrates that the inner gate opens maximally at low pH regardless of the magnitude of the single-channel-open probability, implying that stationary gating originates mostly from rearrangements at the selectivity filter. Two E71A crystal structures obtained at 2.5 A reveal large structural excursions of the selectivity filter during ion conduction and provide a glimpse of the range of conformations available to this region of the channel during gating. These data establish a mechanistic basis for the role of the selectivity filter during channel activation and inactivation.     

Lipids in the structure,folding, and function of the KcsA K+ channel

Lipid molecules surround an ion channel in its native environment of cellular membranes. The importance of the lipid bilayer and the role of lipid protein interactions in ion channel structure and function are not well understood. Here we demonstrate that the bacterial potassium channel KcsA binds a negatively charged lipid molecule. We have defined the potential binding site of the lipid molecule on KcsA by X-ray crystallographic analysis of a complex of KcsA with a monoclonal antibody Fab fragment. We also demonstrate that lipids are required for the in vitro refolding of the KcsA tetramer from the unfolded monomeric state. The correct refolding of the KcsA tetramer requires lipids, but it is not dependent on negatively charged lipids as refolding takes place in the absence of such lipids. We confirm that the presence of negatively charged lipids is required for ion conduction through the KcsA potassium channel, suggesting that the lipid bound to KcsA is important for ion channel function.     

Ion binding to KcsA: implications in ion selectivity and channel gating

Binding of K+ and Na+ to the potassium channel KcsA has been characterized from the stabilization observed in the heat-induced denaturation of the protein as the ion concentration is increased. KcsA thermal denaturation is known to include (i) dissociation of the homotetrameric channel into its constituent subunits and (ii) protein unfolding. The ion concentration-dependent changes in the thermal stability of the protein, evaluated as the Tm value for thermal-induced denaturation of the protein, may suggest the existence of both high- and low-affinity K+ binding sites of KcsA, which lend support to the tenet that channel gating may be governed by K+ concentration-dependent transitions between different affinity states of the channel selectivity filter. We also found that Na+ binds to KcsA with a KD similar to that estimated electrophysiologically from channel blockade. Therefore, our findings on ion binding to KcsA partly account for K+ over Na+ selectivity and Na+ blockade and argue against the strict “snug fit” hypothesis used initially to explain ion selectivity from the X-ray channel structure. Furthermore, the remarkable effects of increasing the ion concentration, K+ in particular, on the Tm of the denaturation process evidence that synergistic effects of the metal-mediated intersubunit interactions at the channel selectivity filter are a major contributor to the stability of the tetrameric protein. This observation substantiates the notion of a role for ions as structural “effectors” of ion channels.     

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" peptide: mapping the inactivating peptide-binding epitope

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.     

Side-chain ionization states in a potassium channel

KcsA is a bacterial K+ channel that is gated by pH. Continuum dielectric calculations on the crystal structure of the channel protein embedded in a low dielectric slab suggest that side chains E71 and D80 of each subunit, which lie adjacent to the selectivity filter region of the channel, form a proton-sharing pair in which E71 is neutral (protonated) and D80 is negatively charged at pH 7. When K+ ions are introduced into the system at their crystallographic positions the pattern of proton sharing is altered. The largest perturbation is for a K+ ion at site S3, i.e., interacting with the carbonyls of T75 and V76. The presence of multiple K+ ions in the filter increases the probability of E71 being ionized and of D80 remaining neutral (i.e., protonated). The ionization states of the protein side chains influence the potential energy profile experienced by a K+ ion as it is translated along the pore axis. In particular, the ionization state of the E71-D80 proton-sharing pair modulates the shape of the potential profile in the vicinity of the selectivity filter. Such reciprocal effects of ion occupancy on side-chain ionization states, and of side-chain ionization states on ion potential energy profiles will complicate molecular dynamics simulations and related studies designed to calculate ion permeation energetics.     

A multipoint hydrogen-bond network underlying KcsA C-type inactivation

In the prokaryotic potassium channel KcsA activation gating at the inner bundle gate is followed by C-type inactivation at the selectivity filter. Entry into the C-type inactivated state has been directly linked to the strength of the H-bond interaction between residues Glu-71 and Asp-80 behind the filter, and is allosterically triggered by the rearrangement of the inner bundle gate. Here, we show that H-bond pairing between residues Trp-67 and Asp-80, conserved in most K⁺ channels, constitutes another critical interaction that determines the rate and extent of KcsA C-type inactivation. Disruption of the equivalent interaction in Shaker (Trp-434-Asp-447) and Kv1.2 (Trp-366-Asp-379) leads also to modulation of the inactivation process, suggesting that these residues also play an analogous role in the inactivation gating of Kv channels. The present results show that in KcsA C-type inactivation gating is governed by a multipoint hydrogen-bond network formed by the triad Trp-67-Glu71-Asp-80. This triad exerts a critical role in the dynamics and conformational stability of the selectivity filter and might serve as a general modulator of selectivity filter gating in other members of the K⁺ channel family.     

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.     

The ionization state and the conformation of Glu-71 in the KcsA K(+) channel.

The side chain of Glu-71 of the KcsA K(+) channel, an important residue in the vicinity of the selectivity filter, was not resolved in the crystallographic structure of Doyle et al. (Doyle, D. A., J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon. 1998. Science. 280:69-77). Its atomic coordinates are undetermined and its ionization state is unknown. For meaningful theoretical and computational studies of the KcsA K(+) channel, it is essential to address questions about the conformation and the ionization state of this residue in detail. In previous MD simulations in which the side chain of Glu-71 is protonated and forming a strong hydrogen bond with Asp-80 it was observed that the channel did not deviate significantly from the crystallographic structure (Bernèche, S., and B. Roux. 2000. Biophys. J. 78:2900-2917). In contrast, we show here that the structure of the selectivity filter of the KcsA channel is significantly disrupted when these side chains are fully ionized on each of the four monomers. To further resolve questions about the ionization state of Glu-71 we calculated the pK(a) value of this residue using molecular dynamics free energy simulations (MD/FES) with a fully flexible system including explicit solvent and membrane and finite-difference Poisson-Boltzmann (PB) continuum electrostatics. It is found that the pK(a) of Glu-71 is shifted by approximately +10 pK(a) units. These results strongly suggest that Glu-71 is protonated under normal conditions.     

Influence of interionic interactions on functional state and blocker binding of voltage-gated potassium channels

A mechanism of ion conduction of a voltage-gated potassium channel KcsA was investigated in full-atomic approximation at a trajectory length of 100 ns using the Lomonosov supercomputer. Methods of molecular dynamics were employed. A structure of the KcsA channel in the open state obtained by X-ray structure analysis (PDB ID 3fb7) was used. Free energy profiles of the KcsA pore occupied with either one or three potassium ions were calculated. It was shown that, under physiological conditions, ions pass through the channel pore cooperatively and the mechanism most probably includes three ions permeating in concert. Interactions of the mammalian voltage-gated channel Kv1.2 with neurotoxin were investigated. It was demonstrated that the effect of interionic interactions on binding of a blocker is rather insufficient.     

Voltage-dependent gating at the KcsA selectivity filter

The prokaryotic K(+) channel KcsA, although lacking a 'standard' voltage-sensing domain, shows voltage-dependent gating that leads to an increase in steady-state open probability of almost two orders of magnitude between +150 and -150 mV. Here we show that voltage-dependent gating in KcsA is associated with the movement of approximately 0.7 equivalent electronic charges. This charge movement produces an increase in the rate of entry into a long-lived inactivated state and seems to be independent of the proton-activation mechanism. Charge neutralization at position 71 renders the channel essentially voltage-independent by preventing entry into the inactivated state. A mechanism for voltage-dependent gating at the selectivity filter is proposed that is based on the reorientation of the carboxylic moiety of Glu71 and its influence in the conformational dynamics of the selectivity filter.     

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites.Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.     

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

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