The permeability of the sodium channel in Myxicola to the alkali cations

Relative permeabilities to the alkali cations were determined, from the reversal potential (VRev), for the Na channel of internally perfused voltage-clamped Myxicola giant axons. PLi/PNa and PK/PNa are 0.94 and 0.076, respectively. Rb and Cs are not measurably permeant. VRev vs. the internal Na activity was well described by the constant field equation over a 300-fold range of internal Na concentrations. In agreement with findings on squid axons, the PK/PNa was found to increase when the K content of the internal perfusate was reduced (equivalent per equivalent substitution with TMA). Internal Rb and Cs also decreased the PK/PNa. The order of effectiveness of internal K, Rb, and Cs in increasing the Na selectivity of the Na channel was Cs greater than Rb greater than or equal to K. External Li increases the PK/PNa but this may be due to the formation of LiF internally. It may be that substances do not have to traverse the channel in order to affect the selectivity filter. Evidence is presented which suggests that the selectivity of the Na channel may be higher for Na in intact as compared to perfused giant axons. It was concluded that the channel selectivity properities do not reflect only some fixed structural features of the channel, but the selectivity filter has a labile organization.     

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.     

Ion selectivity of epithelial Na channels

Epithelial Na channels are apparently pore-forming membrane proteins which conduct Na much better than any other biologically abundant ion. The conductance to Na can be 100 to 1000 times higher than that to K. The only other ions that can readily get through this channel are protons and Li. Small organic cations cannot pass through the channel, and water may also be impermeant. The selectivity properties of epithelial Na channels appear to be determined by at least three factors: A high field-strength anionic site, most likely a carboxyl residue of glutamic or aspartic acid residues on the channel protein, probably accounts for the high conductance through these channels of Na and Li and to the low conductance of K, Rb and Cs. A restriction in the size of the pore at its narrowest point probably accounts for the low conductance of organic cations as well as the possible exclusion of water molecules. The outer mouth of the channel appears to be negatively charged and may control access to the region of highest selectivity and may serve as a preliminary selectivity filter, attracting cations over anions. These conclusions are illustrated by the cartoon of the channel in Fig. 3. This picture is obviously both fanciful and simplified, but its general points will hopefully be testable. It leaves open a number of important questions, including: does amiloride block the channel by binding within the outer mouth? what does the inner mouth of the channel look like, and does this part of the channel contribute to selectivity? and what, if any, are the interactions between the features of the channel that impart selectivity and those that control the regulation of the channel by hormonal and other factors?     

Simulation of voltage-driven hydrated cation transport through narrow transmembrane channels.

Molecular dynamics studies for the voltage-driven transport of the alkali metal ions lithium, sodium, and potassium through gramicidin A-type channels filled with water molecules are presented. The number of water molecules in the channel is obtained from a previous study (Skerra, A., and J. Brickmann, 1987, Biophys. J., 51:969-976). It is shown that the selectivity of the intrachannel ion diffusion through our model pore conforms to the experimentally observed selectivity of the gramicidin A channel. It is demonstrated that the number of water molecules in the channel plays a key role for the selectivity.     

The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel

The Kir3.1/Kir3.4 channel is activated by Gbetagamma subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel.     

Molecular basis of ion selectivity, block, and rectification of the inward rectifier Kir3.1/Kir3.4 K(+) channel

The glycine-tyrosine-glycine (GYG) sequence in the p-loop of K+ channel subunits lines a narrow pore through which K+ ions pass in single file intercalated by water molecules. Mutation of the motif can give rise to non-selective channels, but it is clear that other structural features are also required for selectivity because, for instance, a recently identified class of cyclic nucleotide-gated pacemaker channels has the GYG motif but are poorly K+ selective. We show that mutation of charged glutamate and arginine residues behind the selectivity filter in the Kir3.1/Kir3.4 K+ channel reduces or abolishes K+ selectivity, comparable with previously reported effects in the Kir2.1 K+ channel. It has been suggested that a salt bridge exists between the glutamate-arginine residue pair. Molecular modeling indicates that the salt bridge does exist, and that it acts as a "bowstring" to maintain the rigid bow-like structure of the selectivity filter and restrict selectivity to K+. The modeling shows that relaxation of the bowstring by mutation of the residue pair leads to enhanced flexibility of the p-loop, allowing permeation of other cations, including polyamines. In experiments, mutation of the residue pair can also abolish polyamine-induced inward rectification. The latter effect occurs because polyamines now permeate rather than block the channel, to the remarkable extent that large polyamine currents can be measured.     

Ion solvation and structural stability in a sodium channel investigated by molecular dynamics calculations

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na(+) ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K(+) ions in potassium channels. Our results also indicate that Na(+) ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na(+) permeation through the selectivity filter.     

Colicin N forms voltage- and pH-dependent channels in planar lipid bilayer membranes

The protein antibiotic colicin N forms ion-permeable channels through planar lipid bilayers. Channels are induced when positive voltages higher than +60 mV are applied. Incorporated channels activate and inactivate in a voltage-dependent fashion. It is shown that colicin N undergoes a transition between an “acidic” and a “basic” channel form which are distinguishable by different voltage dependences. The single-channel conductance is non-ohmic and strongly dependent on pH, indicating that titratable groups control the passage of ions through the channel. The ion selectivity of colicin N channels is influenced by the pH and the lipid composition of the bilayer membrane. In neutral membranes the channel undergoes a transition from slightly cation-selective to slightly anion-selective when the pH is changed from 7 to 5. In lipid membranes bearing a negative surface charge the channel shows a more pronounced cation selectivity which decreases but does not reverse upon lowering the pH from 7 to 5. The high degree of similarity between the channel characteristics of colicin A and N suggests that the channels share common features in their molecular structure. Offprint requests to: F. Pattus     

Proton conductance by the gramicidin water wire. Model for proton conductance in the F1F0 ATPases?

The gramicidin channel contains a single strand of water molecules associated through hydrogen bonds. Previous work has shown that channels of similar size are formed by association of transmembrane alpha helices of synthetic leucine-serine peptides. Both types of channels translocate protons with considerable selectivity relative to other cations, and it has been proposed that the selectivity arises by proton "hopping" along hydrogen-bonded chains of water, whereas other cations must cross by ordinary diffusion processes. It is possible that a similar mechanism underlies proton transport in the Fo subunit of the F1F0 ATP synthase. Using the gramicidin channel as a model, we have tested whether a single strand of water is kinetically competent to translocate protons at a rate sufficient to support known rates of ATP synthesis. We found that the gramicidin channel saturates at approximately 530 pS of protonic current in 4 M HCl, more than sufficient for typical ATP synthesis rates. It follows that proton diffusion to a putative channel in Fo, rather than the channel itself, may limit ATP synthesis rates.     

Understanding pH-dependent selectivity of alamethicin K18 channels by computer simulation

Alamethicin K18 is a covalently linked alamethicin dimer in which the glutamine residue at position 18 in each helix has been replaced by a lysine residue. As described in previous work, channels formed by this peptide show pH-dependent selectivity. The maximum anion selectivity of the putative octameric conducting state is obtained at pH 7 or lower. Inasmuch as no change in selectivity is seen between pH 7 and pH 3, and because protons are expected to be in equilibrium with the open state of the channel during a selectivity measurement, the channel is believed to be fully charged (i.e., all eight lysines protonated) at pH 7. In an effort to understand how such a highly charged channel structure is stable in membranes and why it is not more selective for anions, we have performed a number of computer simulations of the system. Molecular dynamics simulations of 10 ns each of the octameric bundle in a lipid bilayer environment are presented, with either zero, four, or eight lysines charged in the absence of salt, and with eight lysines charged in the presence of 0.5 M and 1 M KCl. When no salt is present and all lysines are charged, on average 1.9 Cl(-) ions are inside the channel and the channel significantly deforms. With 0.5 M KCl present, 2.9 Cl(-) ions are inside the channel. With 1 M KCl present, four Cl(-) ions are present and the channel maintains a regular structure. Poisson-Boltzmann calculations on models of the octameric channel also predict an average of 2-4 Cl(-) ions near the lysine residues as a function of ionic strength. These counterions lower the apparent charge of the channel, which may underlie the decrease in selectivity observed experimentally with increasing salt concentrations. We suggest that to increase the selectivity of Alm K18 channels, positive charges could be engineered in a narrower part of the channel.     

Resistance of various protein channels to proteolytic degradation

The influence of some proteolytic enzymes on the properties of the ionic channels induced in the lipid bilayer by three different toxic proteins was studied. It was found that pronase and trypsin did not decrease the conductance of the membranes modified by melittin, alpha-staphylotoxin and latrotoxin and changed its cation-anion selectivity. The dependence of melittin-channels selectivity on the medium pH was investigated. It was suggested that the carbonyl groups of peptide bond take part in defining cation selectivity of the melittin channel.     

Importance of hydration and dynamics on the selectivity of the KcsA and NaK channels

Fundamental concepts governing ion selectivity in narrow pores are reviewed and the microscopic factors responsible for the lack of selectivity of the NaK channel, which is structurally similar to the K+-selective KcsA channel, are elucidated on the basis of all-atom molecular dynamics free energy simulations. The results on NaK are contrasted and compared with previous studies of the KcsA channel. Analysis indicates that differences in hydration of the cation in the pore of NaK is at the origin of the lack of selectivity of NaK.     

Energetics of divalent selectivity in a calcium channel: the ryanodine receptor case study

A model of the ryanodine receptor (RyR) calcium channel is used to study the energetics of binding selectivity of Ca²⁺ versus monovalent cations. RyR is a calcium-selective channel with a DDDD locus in the selectivity filter, similar to the EEEE locus of the L-type calcium channel. While the affinity of RyR for Ca²⁺ is in the millimolar range (as opposed to the micromolar range of the L-type channel), the ease of single-channel measurements compared to L-type and its similar selectivity filter make RyR an excellent candidate for studying calcium selectivity. A Poisson-Nernst-Planck/density functional theory model of RyR is used to calculate the energetics of selectivity. Ca²⁺ versus monovalent selectivity is driven by the charge/space competition mechanism in which selectivity arises from a balance of electrostatics and the excluded volume of ions in the crowded selectivity filter. While electrostatic terms dominate the selectivity, the much smaller excluded-volume term also plays a substantial role. In the D4899N and D4938N mutations of RyR that are analyzed, substantial changes in specific components of the chemical potential profiles are found far from the mutation site. These changes result in the significant reduction of Ca²⁺ selectivity found in both theory and experiments.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

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+.     

The interaction of Cl- with a gramicidin-like channel

Molecular dynamics simulations have been used to study the interaction of Cl- with a gramicidin-like channel. The results suggest that there is a high-energy barrier at the entrance of the channel, which would correspond to a permeability 10(-9)-times that of a cation of the same size. This could account for the cationic selectivity of the gramicidin channel and indicates that valence selectivity is kinetically controlled.     

What really prevents proton transport through aquaporin? Charge self-energy versus proton wire proposals

The nature of the control of water/proton selectivity in biological channels is a problem of a fundamental importance. Most studies of this issue have proposed that an interference with the orientational requirements of the so-called proton wire is the source of selectivity. The elucidation of the structures of aquaporins, which have evolved to prevent proton transfer (PT), provided a clear benchmark for exploring the selectivity problem. Previous simulations of this system have not examined, however, the actual issue of PT, but only considered the much simpler task of the transfer of water molecules. Here we take aquaporin as a benchmark and quantify the origin of the water/proton selectivity in this and related systems. This is done by evaluating in a consistent way the free energy profile for transferring a proton along the channel and relating this profile to the relevant PT rate constants. It is found that the water/proton selectivity is controlled by the change in solvation free energy upon moving the charged proton from water to the channel. The reason for the focus on the elegant concept of the proton wire and the related Grotthuss-type mechanism is also considered. It is concluded that these mechanisms are clearly important in cases with flat free energy surfaces (e.g., in bulk water, in gas phase water chains, and in infinitely long channels). However, in cases of biological channels, the actual PT mechanism is much less important than the energetics of transferring the proton charge from water to different regions in the channels.