Ion channels of glutamate receptors: structural modeling

Ionotropic glutamate receptors belong to the superfamily of P-loop channels as well as K(+), Na(+), and Ca(2+) channels. However, the structural similarity between ion channels of the glutamate receptors and K(+) channels is a matter of discussion. The aim of this study was to analyze differences between the structures of K(+) channels and glutamate receptor channels. For this purpose, homology models of NMDA and AMPA receptor channels (M2 and M3 segments) were built using X-ray structures of K(+) channels as templates. The models were optimized and used to reproduce specific data on the structure of glutamate receptor channels. Particular attention was paid to the data of the binding of channel blockers and to the results of scanning mutagenesis. The modeling demonstrates that properties of glutamate receptor channel can be reproduced assuming only local structural deformations of the K(+) channel templates. The most valuable differences were found in the selectivity-filter region, whereas helical parts of M2 and M3 segments could have similar spatial organization with homologous segments in K(+) channels. It is concluded that the current experimental data on glutamate receptor channels does not reveal global structural differences with K(+) channels.     

Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Molecular insights into the structure and function of plant K(+) transport mechanisms

Our understanding of plant potassium transport has increased in the past decade through the application of molecular biological techniques. In this review, recent work on inward and outward rectifying K(+) channels as well as high affinity K(+) transporters is described. Through the work on inward rectifying K(+) channels, we now have precise details on how the structure of these proteins determines functional characteristics such as ion conduction, pH sensitivity, selectivity and voltage sensing. The physiological function of inward rectifying K(+) channels in plants has been clarified through the analysis of expression patterns and mutational analysis. Two classes of outward rectifying K(+) channels have now been cloned from plants and their initial characterisation is reviewed. The physiological role of one class of outward rectifying K(+) channel has been demonstrated to be involved in long distance transport of K(+) from roots to shoots. The molecular structure and function of two classes of energised K(+) transporters are also reviewed. The first class is energised by Na(+) and shares structural similarities with K(+) transport mechanisms in bacteria and fungi. Structure-function studies suggest that it should be possible to increase the K(+) and Na(+) selectivity of these transporters, which will enhance the salt tolerance of higher plants. The second class of K(+) transporter is comprised of a large gene family and appears to have a dual affinity for K(+). A suite of molecular techniques, including gene cloning, oocyte expression, RNA localisation and gene inactivation, is now being used to fully characterise the biophysical and physiological function of plants K(+) transport mechanisms.     

A pore segment in DEG/ENaC Na(+) channels.

DEG/ENaC Na(+) channels have diverse functions, including Na(+) absorption, neurotransmission, and sensory transduction. The ability of these channels to discriminate between different ions is critical for their normal function. Several findings suggest that DEG/ENaC channels have a pore structure similar to K(+) channels. To test this hypothesis, we examined the accessibility of native and introduced cysteines in the putative P loop of ENaC. We identified residues that span a barrier that excludes amiloride as well as anionic and large methanethiosulfonate reagents from the pore. This segment contains a structural element ((S/G)CS) involved in selectivity of ENaC. The results are not consistent with predictions from the K(+) channel pore, suggesting that DEG/ENaC Na(+) channels have a novel pore structure.     

KappaM-conotoxin RIIIK, structural and functional novelty in a K+ channel antagonist

Venomous organisms have evolved a variety of structurally diverse peptide neurotoxins that target ion channels. Despite the lack of any obvious structural homology, unrelated toxins that interact with voltage-activated K(+) channels share a dyad motif composed of a lysine and a hydrophobic amino acid residue, usually a phenylalanine or a tyrosine. kappaM-Conotoxin RIIIK (kappaM-RIIIK), recently characterized from the cone snail Conus radiatus, blocks Shaker and TSha1 K(+) channels. The functional and structural study presented here reveals that kappaM-conotoxin RIIIK blocks voltage-activated K(+) channels with a novel pharmacophore that does not comprise a dyad motif. Despite the quite different amino acid sequence and no overlap in the pharmacological activity, we found that the NMR solution structure of kappaM-RIIIK in the C-terminal half is highly similar to that of mu-conotoxin GIIIA, a specific blocker of the skeletal muscle Na(+) channel Na(v)1.4. Alanine substitutions of all non-cysteine residues indicated that four amino acids of kappaM-RIIIK (Leu1, Arg10, Lys18, and Arg19) are key determinants for interaction with K(+) channels. Following the hypothesis that Leu1, the major hydrophobic amino acid determinant for binding, serves as the hydrophobic partner of a dyad motif, we investigated the effect of several mutations of Leu1 on the biological function of kappaM-RIIIK. Surprisingly, both the structural and mutational analysis suggested that, uniquely among well-characterized K(+) channel-targeted toxins, kappaM-RIIIK blocks voltage-gated K(+) channels with a pharmacophore that is not organized around a lysine-hydrophobic amino acid dyad motif.     

A homology model of the pore region of HCN channels

HCN channels are activated by membrane hyperpolarization and regulated by cyclic nucleotides, such as cyclic adenosine-mono-phosphate (cAMP). Here we present structural models of the pore region of these channels obtained by using homology modeling and validated against spatial constraints derived from electrophysiological experiments. For the construction of the models we make two major assumptions, justified by electrophysiological observations: i), in the closed state, the topology of the inner pore of HCN channels is similar to that of K(+) channels. In particular, the orientation of the S5 and S6 helices of HCN channels is very similar to that of the corresponding helices of the K(+) KcsA and K(+) KirBac1.1 channels. Thus, we use as templates the x-ray structure of these K(+) channels. ii), In the open state, the S6 helix is bent further than it is in the closed state, as suggested (but not proven) by experimental data. For this reason, the template of the open conformation is the x-ray structure of the MthK channel. The structural models of the closed state turn out to be consistent with all the available electrophysiological data. The model of the open state turned out to be consistent with all the available electrophysiological data in the filter region, including additional experimental data performed in this work. However, it required the introduction of an appropriate, experimentally derived constraint for the S6 helix. Our modeling provides a structural framework for understanding several functional properties of HCN channels: i), the cysteine ring at the inner mouth of the pore may act as a sensor of the intracellular oxidizing/reducing conditions; ii), the bending amplitude of the S6 helix upon gating appears to be significantly smaller than that found in MthK channels; iii), the reduced ionic selectivity of HCN channels, relative to that of K(+) channels, may be caused, at least in part, by the larger flexibility of the inner pore of HCN channels.     

Sequence-function analysis of the K+-selective family of ion channels using a comprehensive alignment and the KcsA channel structure

Sequence-function analysis of K(+)-selective channels was carried out in the context of the 3.2 A crystal structure of a K(+) channel (KcsA) from Streptomyces lividans (Doyle et al., 1998). The first step was the construction of an alignment of a comprehensive set of K(+)-selective channel sequences forming the putative permeation path. This pathway consists of two transmembrane segments plus an extracellular linker. Included in the alignment are channels from the eight major classes of K(+)-selective channels from a wide variety of species, displaying varied rectification, gating, and activation properties. Segments of the alignment were assigned to structural motifs based on the KcsA structure. The alignment's accuracy was verified by two observations on these motifs: 1), the most variability is shown in the turret region, which functionally is strongly implicated in susceptibility to toxin binding; and 2), the selectivity filter and pore helix are the most highly conserved regions. This alignment combined with the KcsA structure was used to assess whether clusters of contiguous residues linked by hydrophobic or electrostatic interactions in KcsA are conserved in the K(+)-selective channel family. Analysis of sequence conservation patterns in the alignment suggests that a cluster of conserved residues is critical for determining the degree of K(+) selectivity. The alignment also supports the near-universality of the "glycine hinge" mechanism at the center of the inner helix for opening K channels. This mechanism has been suggested by the recent crystallization of a K channel in the open state. Further, the alignment reveals a second highly conserved glycine near the extracellular end of the inner helix, which may be important in minimizing deformation of the extracellular vestibule as the channel opens. These and other sequence-function relationships found in this analysis suggest that much of the permeation path architecture in KcsA is present in most K(+)-selective channels. Because of this finding, the alignment provides a robust starting point for homology modeling of the permeation paths of other K(+)-selective channel classes and elucidation of sequence-function relationships therein. To assay these applications, a homology model of the Shaker A channel permeation path was constructed using the alignment and KcsA as the template, and its structure evaluated in light of established structural criteria.     

Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution

Inward rectifier K(+) channels govern the resting membrane voltage in many cells. Regulation of these ion channels via G protein-coupled receptor signaling underlies the control of heart rate and the actions of neurotransmitters in the central nervous system. We have determined the protein structure formed by the intracellular N- and C termini of the G protein-gated inward rectifier K(+) channel GIRK1 at 1.8 A resolution. A cytoplasmic pore, conserved among inward rectifier K(+) channels, extends the ion pathway to 60 A, nearly twice the length of a canonical transmembrane K(+) channel. The cytoplasmic pore is lined by acidic and hydrophobic amino acids, creating a favorable environment for polyamines, which block the pore. These results explain in structural and chemical terms the basis of inward rectification, and they also have implications for G protein regulation of GIRK channels.     

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.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.     

The contribution of hydrophobic residues in the pore-forming region of the ryanodine receptor channel to block by large tetraalkylammonium cations and Shaker B inactivation peptides

Although no high-resolution structural information is available for the ryanodine receptor (RyR) channel pore-forming region (PFR), molecular modeling has revealed broad structural similarities between this region and the equivalent region of K(+) channels. This study predicts that, as is the case in K(+) channels, RyR has a cytosolic vestibule lined with predominantly hydrophobic residues of transmembrane helices (TM10). In K(+) channels, this vestibule is the binding site for blocking tetraalkylammonium (TAA) cations and Shaker B inactivation peptides (ShBPs), which are stabilized by hydrophobic interactions involving specific residues of the lining helices. We have tested the hypothesis that the cytosolic vestibule of RyR fulfils a similar role and that TAAs and ShBPs are stabilized by hydrophobic interactions with residues of TM10. Both TAAs and ShBPs block RyR from the cytosolic side of the channel. By varying the composition of TAAs and ShBPs, we demonstrate that the affinity of both species is determined by their hydrophobicity, with variations reflecting alterations in the dissociation rate of the bound blockers. We investigated the role of TM10 residues of RyR by monitoring block by TAAs and ShBPs in channels in which the hydrophobicity of individual TM10 residues was lowered by alanine substitution. Although substitutions changed the kinetics of TAA interaction, they produced no significant changes in ShBP kinetics, indicating the absence of specific hydrophobic sites of interactions between RyR and these peptides. Our investigations (a) provide significant new information on both the mechanisms and structural components of the RyR PFR involved in block by TAAs and ShBPs, (b) highlight important differences in the mechanisms and structures determining TAA and ShBP block in RyR and K(+) channels, and (c) demonstrate that although the PFRs of these channels contain analogous structural components, significant differences in structure determine the distinct ion-handling properties of the two species of channel.     

Binding of kappa-conotoxin PVIIA to Shaker K+ channels reveals different K+ and Rb+ occupancies within the ion channel pore

The x-ray structure of the KcsA channel at different [K(+)] and [Rb(+)] provided insight into how K(+) channels might achieve high selectivity and high K(+) transit rates and showed marked differences between the occupancies of the two ions within the ion channel pore. In this study, the binding of kappa-conotoxin PVIIA (kappa-PVIIA) to Shaker K(+) channel in the presence of K(+) and Rb(+) was investigated. It is demonstrated that the complex results obtained were largely rationalized by differences in selectivity filter occupancy of this 6TM channels as predicted from the structural work on KcsA. kappa-PVIIA inhibition of the Shaker K(+) channel differs in the closed and open state. When K(+) is the only permeant ion, increasing extracellular [K(+)] decreases kappa-PVIIA affinity for closed channels by decreasing the "on" binding rate, but has no effect on the block of open channels, which is influenced only by the intracellular [K(+)]. In contrast, extracellular [Rb(+)] affects both closed- and open-channel binding. As extracellular [Rb(+)] increases, (a) binding to the closed channel is slightly destabilized and acquires faster kinetics, and (b) open channel block is also destabilized and the lowest block seems to occur when the pore is likely filled only by Rb(+). These results suggest that the nature of the permeant ions determines both the occupancy and the location of the pore site from which they interact with kappa-PVIIA binding. Thus, our results suggest that the permeant ion(s) within a channel pore can determine its functional and pharmacological properties.     

Crystallographic study of the tetrabutylammonium block to the KcsA K+ channel

K(+) channels play essential roles in regulating membrane excitability of many diverse cell types by selectively conducting K(+) ions through their pores. Many diverse molecules can plug the pore and modulate the K(+) current. Quaternary ammonium (QA) ions are a class of pore blockers that have been used for decades by biophysicists to probe the pore, leading to important insights into the structure-function relation of K(+) channels. However, many key aspects of the QA-blocking mechanisms remain unclear to date, and understanding these questions requires high resolution structural information. Here, we address the question of whether intracellular QA blockade causes conformational changes of the K(+) channel selectivity filter. We have solved the structures of the KcsA K(+) channel in complex with tetrabutylammonium (TBA) and tetrabutylantimony (TBSb) under various ionic conditions. Our results demonstrate that binding of TBA or TBSb causes no significant change in the KcsA structure at high concentrations of permeant ions. We did observe the expected conformational change of the filter at low concentration of K(+), but this change appears to be independent of TBA or TBSb blockade.     

Structural understanding of the transmembrane domains of inositol triphosphate receptors and ryanodine receptors towards calcium channeling.

Inositol 1,4,5-triphosphate receptors (Insp(3)Rs) and ryanodine receptors (ryRs) act as cationic channels transporting calcium ions from the endoplasmic reticulum to cytosol by forming tetramers and are proteins localized to the endoplasmic reticulum (ER). Despite the absence of classical calcium-binding motifs, calcium channeling occurs at the transmembrane domain. We have investigated putative calcium binding motifs in these sequences. Prediction methods indicate the presence of six transmembrane helices in the C-terminal domain, one of the three domains conserved between Insp(3)R and ryR receptors. The recently identified crystal structure of the K(+) channel, which also forms tetramers, revealed that two transmembrane helices, an additional pore helix and a selectivity filter are responsible for selective K(+) ion channeling. The last three TM helices of Insp(3)R and ryR are particularly well conserved and we found analogous pore helix and selectivity filter motif in these sequences. We obtained a three-dimensional structural model for the transmembrane tetramer by extrapolating the distant structural similarity to the K(+) channels.     

Basolateral membrane K+ channels in renal epithelial cells

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K(+) channels play critical roles in normal physiology. Over 90 different genes for K(+) channels have been identified in the human genome. Epithelial K(+) channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K(+) channels is to recycle K(+) across the basolateral membrane for proper function of the Na(+)-K(+)-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K(+) channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a "K(+) channel gene family" approach in presenting the representative basolateral K(+) channels of the nephron. The basolateral K(+) channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families.     

Critical assessment of a proposed model of Shaker

Detailed three-dimensional structures at atomic resolution are essential to understand how voltage-activated K(+) channels function. The X-ray crystallographic structure of the KvAP channel has offered the first view at atomic resolution of the molecular architecture of a voltage-activated K(+) channel. In the crystal, the voltage sensors are bound by monoclonal Fab fragments, which apparently induce a non-native conformation of the tetrameric channel. Thus, despite this significant advance our knowledge of the native conformation of a Kv channel in a membrane remains incomplete. Numerous results from different experimental approaches provide very specific constraints on the structure of K(+) channels in functional conformations. These results can be used to go further in trying to picture the native conformation of voltage-gated K(+) channels. However, the direct translation of all the available information into three-dimensional models is not straightforward and many questions about the structure of voltage-activated K(+) channels are still unanswered. Our aim in this review is to summarize the most important pieces of information currently available and to provide a critical assessment of the model of Shaker recently proposed by Lainé et al.     

State-dependent block of BK channels by synthesized shaker ball peptides

Crystal structures of potassium channels have strongly corroborated an earlier hypothetical picture based on functional studies, in which the channel gate was located on the cytoplasmic side of the pore. However, accessibility studies on several types of ligand-sensitive K(+) channels have suggested that their activation gates may be located near or within the selectivity filter instead. It remains to be determined to what extent the physical location of the gate is conserved across the large K(+) channel family. Direct evidence about the location of the gate in large conductance calcium-activated K(+) (BK) channels, which are gated by both voltage and ligand (calcium), has been scarce. Our earlier kinetic measurements of the block of BK channels by internal quaternary ammonium ions have raised the possibility that they may lack a cytoplasmic gate. We show in this study that a synthesized Shaker ball peptide (ShBP) homologue acts as a state-dependent blocker for BK channels when applied internally, suggesting a widening at the intracellular end of the channel pore upon gating. This is consistent with a gating-related conformational change at the cytoplasmic end of the pore-lining helices, as suggested by previous functional and structural studies on other K(+) channels. Furthermore, our results from two BK channel mutations demonstrate that similar types of interactions between ball peptides and channels are shared by BK and other K(+) channel types.     

Tethering chemistry and K+ channels

Voltage-gated K(+) channels are dynamic macromolecular machines that open and close in response to changes in membrane potential. These multisubunit membrane-embedded proteins are responsible for governing neuronal excitability, maintaining cardiac rhythmicity, and regulating epithelial electrolyte homeostasis. High resolution crystal structures have provided snapshots of K(+) channels caught in different states with incriminating molecular detail. Nonetheless, the connection between these static images and the specific trajectories of K(+) channel movements is still being resolved by biochemical experimentation. Electrophysiological recordings in the presence of chemical modifying reagents have been a staple in ion channel structure/function studies during both the pre- and post-crystal structure eras. Small molecule tethering agents (chemoselective electrophiles linked to ligands) have proven to be particularly useful tools for defining the architecture and motions of K(+) channels. This Minireview examines the synthesis and utilization of chemical tethering agents to probe and manipulate the assembly, structure, function, and molecular movements of voltage-gated K(+) channel protein complexes.     

Cytoplasmic gatekeepers of K+-channel flux: a structural perspective

Recently, rapid progress in our structural knowledge of K(+)-selective channels has started to provide a basis for comprehending the biophysical machinery underlying their electrophysiological properties. These studies have begun to reveal how a diverse array of distinct, cytoplasmically positioned domains affect the activity of associated channels. Some of these establish functional diversity by selectively mediating channel assembly. More importantly, these cytoplasmic domains couple intracellular signals to the gating of their associated pore. New structural insights are providing a clearer understanding of the fundamental molecular mechanisms of these K(+) channels that, in turn, partly underlie complex neurological phenomena.