KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

The KdpC subunit of the Escherichia coli K+-transporting KdpB P-type ATPase acts as a catalytic chaperone

In Bacteria and Archaea, high-affinity potassium uptake is mediated by the ATP-driven KdpFABC complex. On the basis of the biochemical properties of the ATP-hydrolyzing subunit KdpB, the transport complex is classified as type IA P-type ATPase. However, the KdpA subunit, which promotes K(+) transport, clearly resembles a potassium channel, such that the KdpFABC complex represents a chimera of ion pumps and ion channels. In the present study, we demonstrate that the blending of these two groups of transporters in KdpFABC also entails a nucleotide-binding mechanism in which the KdpC subunit acts as a catalytic chaperone. This mechanism is found neither in P-type ATPases nor in ion channels, although parallels are found in ABC transporters. In the latter, the ATP nucleotide is coordinated by the LSGGQ signature motif via double hydrogen bonds at a conserved glutamine residue, which is also present in KdpC. High-affinity nucleotide binding to the KdpFABC complex was dependent on the presence of this conserved glutamine residue in KdpC. In addition, both ATP binding to KdpC and ATP hydrolysis activity of KdpFABC were sensitive to the accessibility, presence or absence of the hydroxyl groups at the ribose moiety of the nucleotide. Furthermore, the KdpC subunit was shown to interact with the nucleotide-binding loop of KdpB in an ATP-dependent manner around the ATP-binding pocket, thereby increasing the ATP-binding affinity by the formation of a transient KdpB/KdpC/ATP ternary complex.     

FITC binding site and p-nitrophenyl phosphatase activity of the Kdp-ATPase of Escherichia coli

The KdpFABC complex of Escherichia coli, which belongs to the P-type ATPase family, has a unique structure, since catalytic activity (KdpB) and the capacity to transport potassium ions (KdpA) are located on different subunits. We found that fluorescein 5-isothiocyanate (FITC) inhibits ATPase activity, probably by covalently modifying lysine 395 in KdpB. In addition, we observed that the KdpFABC complex is able to hydrolyze p-nitrophenyl phosphate (pNPP) in a Mg(2+)-dependent reaction. The pNPPase activity is inhibited by FITC and o-vanadate. Low concentrations of ATP (1-30 microM) stimulate the pNPPase activity, while concentrations of >500 microM are inhibitory. This behavior can be explained either by a regulatory ATP binding site, where ATP hydrolysis is required, or by proposing an interactive dimer. The notion that FITC inhibits pNPPase and ATPase activity supports the idea that the catalytic domain of KdpB is much more compact than other P-type ATPases, like Na(+),K(+)-ATPase, H(+),K(+)-ATPase, and Ca(2+)-ATPase.     

Amino acid substitutions in putative selectivity filter regions III and IV in KdpA alter ion selectivity of the KdpFABC complex from Escherichia coli

When grown under conditions of potassium limitation or high osmolality, Escherichia coli synthesizes the K(+)-translocating KdpFABC complex. The KdpA subunit, which has sequence homology to potassium channels of the KcsA type, has been shown to be important for potassium binding and transport. Replacement of the glycine residues in KdpA at positions 345 and 470, members of putative selectivity filter regions III and IV, alters the ion selectivity of the KdpFABC complex.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

The synaptic vesicle protein synaptophysin: purification and characterization of its channel activity

The synaptic vesicle protein synaptophysin was solubilized from rat brain synaptosomes with a relatively low concentration of Triton X-100 (0.2%) and was highly purified (above 95%) using a rapid single chromatography step on hydroxyapatite/celite resin. Purified synaptophysin was reconstituted into a planar lipid bilayer and the channel activity of synaptophysin was characterized. In asymmetric KCl solutions (cis 300 mM/trans 100 mM), synaptophysin formed a fast-fluctuating channel with a conductance of 414 +/- 13 pS at +60 mV. The open probability of synaptophysin channels was decreased upon depolarization, and channels were found to be cation-selective. Synaptophysin channels showed higher selectivity for K(+) over Cl(-) (P(K(+))/P(Cl(-)) > 8) and preferred K(+) over Li(+), Na(+), Rb(+), Cs(+), or choline(+). The synaptophysin channel is impermeable to Ca(2+), which has no effect on its channel activity. This study is the second demonstration of purified synaptophysin channel activity, but the first biophysical characterization of its channel properties. The availability of large amounts of purified synaptophysin and of its characteristic channel properties might help to establish the role of synaptophysin in synaptic transmission.     

RGD-CAP ((beta)ig-h3) is expressed in precartilage condensation and in prehypertrophic chondrocytes during cartilage development

The TRK-HKT family of K(+) transporters mediates K(+) and Na(+) uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K(+) or Rb(+), both of which bind the two binding sites of ScTRK1 with very high affinities in K(+)-starved cells. Na(+) can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K(+) and Na(+), or Rb(+) and Na(+), the transporter takes up Na(+) together with K(+) or Rb(+). Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln(90) is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu(949) is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K(+) uptake. This is not Na(+)-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K(+)-H(+) cotransport.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Kinetic and thermodynamic analysis of the interaction of cations with dialkylglycine decarboxylase

Dialkylglycine decarboxylase (DGD) is a tetrameric pyridoxal phosphate (PLP)-dependent enzyme that catalyzes both decarboxylation and transamination in its normal catalytic cycle. Its activity is dependent on cations. Metal-free DGD and DGD complexes with seven monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) and three divalent cations (Mg(2+), Ca(2+), and Ba(2+)) have been studied. The catalytic rate constants for cation-bound enzyme (ck(cat) and ck(cat)/bK(AIB)) are cation-size-dependent, K(+) being the monovalent cation with the optimal size for catalytic activity. The divalent alkaline earth cations (Mg(2+), Ca(2+), and Ba(2+)) all give approximately 10-fold lower activity compared to monovalent alkali cations of similar ionic radius. The Michaelis constant for aminoisobutyrate (AIB) binding to DGD-PLP complexes with cations (bK(AIB)) varies with ionic radius. The larger cations (K(+), Rb(+), Cs(+), NH(4)(+), and Tl(+)) give smaller bK(AIB) ( approximately 4 mM), while smaller cations (Li(+), Na(+)) give larger values (approximately 10 mM). Cation size and charge dependence is also found with the dissociation constant for PLP binding to DGD-cation complexes (aK(PLP)). K(+) and Rb(+) possess the optimal ionic radius, giving the lowest values of aK(PLP). The divalent alkaline earth cations give aK(PLP) values approximately 10-fold higher than alkali cations of similar ionic radius. The cation dissociation constant for DGD-PLP-AIB-cation complexes (betaK(M)z+) was determined and also shown to be cation-size-dependent, K(+) and Rb(+) yielding the lowest values. The kinetics of PLP association and dissociation from metal-free DGD and its complexes with cations (Na(+), K(+), and Ba(2+)) were analyzed. All three cations tested increase PLP association and decrease PLP dissociation rate constants. Kinetic studies of cation binding show saturation kinetics for the association reaction. The half-life for association with saturating Rb(+) is approximately 24 s, while the half-life for dissociation of Rb(+) from the DGD-PLP-AIB-Rb(+) complex is approximately 12 min.     

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme. The functional roles of this third K(+)/Rb(+) binding site were studied by site-directed mutagenesis, replacing the side chain of Asp(742) donating oxygen ligand(s) to the site with alanine, glutamate, and lysine. Unlike the wild-type Na(+), K(+)-ATPase, the mutants display a biphasic K(+) concentration dependence of E(2)P dephosphorylation, indicating that the cytoplasmic K(+) site is involved in activation of dephosphorylation. The affinity of the site is lowered significantly (30-200-fold) by the mutations, the lysine mutation being most disruptive. Moreover, the mutations accelerate the E(2) to E(1) conformational transition, again with the lysine substitution resulting in the largest effect. Hence, occupation of the cytoplasmic K(+)/Rb(+) site not only enhances E(2)P dephosphorylation but also stabilizes the E(2) dephosphoenzyme. These characteristics of the previously unrecognized nontransport site make it possible to account for the hitherto poorly understood trans-effects of cytoplasmic K(+) by the consecutive transport model, without implicating a simultaneous exposure of the transport sites toward the cytoplasmic and extracellular sides of the membrane. The cytoplasmic K(+)/Rb(+) site appears to be conserved among Na(+), K(+)-ATPases and P-type ATPases in general, and its mode of operation may be associated with stabilizing the loop structure at the C-terminal end of the P6 helix of the P-domain, thereby affecting the function of highly conserved catalytic residues and promoting helix-helix interactions between the P- and A-domains in the E(2) state.     

Schiff base of gossypol with 3,6,9-trioxa-decylamine complexes with monovalent cations studied by mass spectrometry, (1)H-NMR, FTIR, and PM5 semiempirical methods

A new Schiff base of gossypol with 3,6,9-trioxo-decylamine (GSTB) forms stable complexes with monovalent cations. This process of complex formation was studied by electrospray ionization mass spectrometry, (1)H-NMR and FTIR spectroscopy, and the PM5 (parametric method 5) semiempirical method. It is found that GSTB forms 1 : 1 and 1 : 2 complexes with Li(+) and Na(+) and 1 : 1 complexes with K(+), Rb(+), or Cs(+) cations and exists in all these complexes in the enamine-enamine tautomeric form. Moreover, within these complexes only Li(+) cations can fluctuate between the oxygen atoms of trioxo-alkyl chains. All other cations are strongly localized. In the complex of GSTB with two protons localized on the N atoms of the Schiff base, the imine-imine tautomeric form is realized. The complexes of the Schiff base with K(+), Rb(+), or Cs(+) cations are the 1 : 1 type with the oxygen atoms of the trioxo-alkyl chains, as well as the O(1)H or O(1')H group coordinating the cation. The structures of the complexes are calculated by the PM5 semiempirical method and discussed.     

Gramicidin, valinomycin, and cation permeability of Streptococcus faecalis

Gramicidin and valinomycin in concentrations of 10(-7) and 10(-6)m, respectively, inhibited the growth of Streptococcus faecalis. Inhibition of growth was associated with loss of Rb(+) and K(+) from the cells, and could be reversed by addition of excess K(+). Cells treated with these antibiotics exhibited greatly increased permeability to certain cations; no effect was observed on the penetration of other small molecules. Unlike normal cells, cells treated with gramicidin rapidly lost internal Rb(+) by passive exchange with external cations, including H(+), all monovalent alkali metals, NH(4) (+), Mg(++), and tris(hydroxymethyl)aminomethane. Exchange was rapid even at 0 C and was independent of energy metabolism. The effect of valinomycin was more selective. Cellular Rb(+) was rapidly displaced by external H(+), K(+), Rb(+), and Cs(+); other cations were less effective. The exchange was independent of metabolism but strongly affected by temperature. Under certain conditions, polyvalent cations inhibited exchange between (86)Rb and Rb(+) induced by valinomycin. The antibiotic apparently neither stimulates nor inhibits the energy-dependent K(+) pump of S. faecalis, but exerts its effect on the passive permeability of the membrane to cations. The increased permeability to specific cations induced by gramicidin and valinomycin is a sufficient explanation for the inhibition of growth, glycolysis, and other processes.     

Effects of nigericin and monactin on cation permeability of Streptococcus faecalis and metabolic capacities of potassium-depleted cells

At a concentration of 10(-6)m, nigericin and monactin inhibited growth of Streptococcus faecalis, and the inhibition was reversed by addition of excess K(+). In the presence of certain antibiotics, the cells exhibited increased permeability to certain cations; internal Rb(+) was rapidly lost by exchange with external H(+), K(+) Rb(+), and, more slowly, with Na(+) and Li(+). No effect was observed on the penetration of other small molecules. Cation exchanges induced by nigericin and monactin were metabolically passive and apparently did not involve the energy-dependent K(+) pump. When the cells were washed, the cytoplasmic membrane recovered its original impermeability to cations. By use of monactin, we prepared cells whose K(+) content had been completely replaced by other cations, and the metabolic characteristics of K(+)-depleted cells were studied. Cells containing only Na(+) glycolyzed almost as well as did normal ones and, under proper conditions, could accumulate amino acids and orthophosphate. These cells also incorporated (14)C-uracil into ribonucleic acid but incorporation of (14)C-leucine into protein was strictly dependent upon the addition of K(+). When K(+) or Rb(+) was added to sodium-loaded cells undergoing glycolysis, these ions were accumulated by stoichiometric exchange for Na(+). From concurrent measurements of the rate of glycolysis, it was calculated that one mole-pair of cations was exchanged for each mole of adenosine triphosphate produced.     

The conserved dipole in transmembrane helix 5 of KdpB in the Escherichia coli KdpFABC P-type ATPase is crucial for coupling and the electrogenic K+-translocation step

The KdpFABC complex of Escherichia coli, a high-affinity K+-uptake system, belongs to the group of P-type ATPases and is responsible for ATP-driven K+ uptake in the case of K+ limitation. Sequence alignments identified two conserved charged residues, D583 and K586, which are located at the center of transmembrane helix 5 (TM 5) of the catalytic KdpB subunit, and which are supposed to establish a dipole involved in energy coupling. Cells in which the two charges were eliminated or inverted by mutagenesis displayed a clearly slower growth rate with respect to wild-type cells under K+-limiting conditions. Purified KdpFABC complexes from several K586 mutants and a D583K:K586D double mutant showed a reduced K+-stimulated ATPase activity together with an increased resistance to orthovanadate. Upon reconstitution into liposomes, only the conservative K586R mutant was able to facilitate K+ transport, whereas the elimination of the positive charge at position 586 as well as inverting the charges at positions 583 and 586 (D583K:K586D) led to an uncoupling of ATP hydrolysis and K+ transport. Electrophysiological measurements with KdpFABC-containing proteoliposomes adsorbed to planar lipid bilayers revealed that in case of the D583K:K586D double mutant the characteristic K+-independent electrogenic step within the reaction cycle is lacking, thereby clearly arguing for an exact positioning of the dipole for coupling within the functional enzyme complex. In addition, these findings strongly suggest that the dipole residues in KdpB are not directly responsible for the characteristic electrogenic reaction step of KdpFABC, which most likely occurs within the K+-translocating KdpA subunit.     

7Li-NMR and FTIR studies of lithium, potassium, rubidium, and cesium complexes with ionophore lasalocid in solution

Lasalocid metal salts were combined with 1 : 1 lithium and 2:2 potassium, rubidium, and cesium to form complexes. The nature of the lasolocid salt complexes was studied in a solid and chloroform by FTIR spectroscopy in the middle and far IR regions. The process of the complexation of lithium was also studied by (7)Li-NMR. In chloroform a 1 : 1 complex of lasalocid and Li(+) ions was formed. Continuous absorption was observed in the far FTIR spectrum of this complex. It indicated large Li(+) polarizability, which was due to fast fluctuations of the Li(+) ions in the multiminima potentials, in the monomeric structure. In the lasalocid salt with the other monovalent cations (K(+), Rb(+), Cs(+)) 2:2 complexes were formed in which the cations showed cation polarizability, which strongly depended on the mass and the radius of the cations.     

Cation transport by the neuronal K(+)-Cl(-) cotransporter KCC2: thermodynamics and kinetics of alternate transport modes

Both Cs(+) and NH(4)(+) alter neuronal Cl(-) homeostasis, yet the mechanisms have not been clearly elucidated. We hypothesized that these two cations altered the operation of the neuronal K(+)-Cl(-) cotransporter (KCC2). Using exogenously expressed KCC2 protein, we first examined the interaction of cations at the transport site of KCC2 by monitoring furosemide-sensitive (86)Rb(+) influx as a function of external Rb(+) concentration at different fixed external cation concentrations (Na(+), Li(+), K(+), Cs(+), and NH(4)(+)). Neither Na(+) nor Li(+) affected furosemide-sensitive (86)Rb(+) influx, indicating their inability to interact at the cation translocation site of KCC2. As expected for an enzyme that accepts Rb(+) and K(+) as alternate substrates, K(+) was a competitive inhibitor of Rb(+) transport by KCC2. Like K(+), both Cs(+) and NH(4)(+) behaved as competitive inhibitors of Rb(+) transport by KCC2, indicating their potential as transport substrates. Using ion chromatography to measure unidirectional Rb(+) and Cs(+) influxes, we determined that although KCC2 was capable of transporting Cs(+), it did so with a lower apparent affinity and maximal velocity compared with Rb(+). To assess NH(4)(+) transport by KCC2, we monitored intracellular pH (pH(i)) with a pH-sensitive fluorescent dye after an NH(4)(+)-induced alkaline load. Cells expressing KCC2 protein recovered pH(i) much more rapidly than untransfected cells, indicating that KCC2 can mediate net NH(4)(+) uptake. Consistent with KCC2-mediated NH(4)(+) transport, pH(i) recovery in KCC2-expressing cells could be inhibited by furosemide (200 microM) or removal of external [Cl(-)]. Thermodynamic and kinetic considerations of KCC2 operating in alternate transport modes can explain altered neuronal Cl(-) homeostasis in the presence of Cs(+) and NH(4)(+).     

Characterization of amino acid substitutions in KdpA,the K+-binding and -translocating subunit of the KdpFABC complex of Escherichia coli

When grown under K+ limitation, Escherichia coli induces the K+-translocating KdpFABC complex. The stimulation of ATPase activity by NH4+ ions was shown for the first time. Substitutions in KdpA, which is responsible for K+ binding and translocation, revealed that enzyme complexes KdpA:G232A and KdpA:G232S have completely lost their cation selectivity.     

Potassium channel, ions, and water: simulation studies based on the high resolution X-ray structure of KcsA

Interactions of Na(+), K(+), Rb(+), and Cs(+) ions within the selectivity filter of a potassium channel have been investigated via multiple molecular dynamics simulations (total simulation time, 48 ns) based on the high resolution structure of KcsA, embedded in a phospholipid bilayer. As in simulations based on a lower resolution structure of KcsA, concerted motions of ions and water within the filter are seen. Despite the use of a higher resolution structure and the inclusion of four buried water molecules thought to stabilize the filter, this region exhibits a significant degree of flexibility. In particular, pronounced distortion of filter occurs if no ions are present within it. The two most readily permeant ions, K(+) and Rb(+), are similar in their interactions with the selectivity filter. In contrast, Na(+) ions tend to distort the filter by binding to a ring of four carbonyl oxygens. The larger Cs(+) ions result in a small degree of expansion of the filter relative to the x-ray structure. Cs(+) ions also appear to interact differently with the gate region of the channel, showing some tendency to bind within a predominantly hydrophobic pocket. The four water molecules buried between the back of the selectivity filter and the remainder of the protein show comparable mobility to the surrounding protein and do not exchange with water molecules within the filter or the central cavity. A preliminary comparison of the use of particle mesh Ewald versus cutoff protocols for the treatment of long-range electrostatics suggests some difference in the kinetics of ion translocation within the filter.