Solution structure of the potassium channel inhibitor agitoxin 2: caliper for probing channel geometry.

The structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple-stranded antiparallel beta-sheet and a single helix covering one face of the beta-sheet. The cysteine side chains connecting the beta-sheet and the helix form the core of the molecule. One edge of the beta-sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface-exposed residues, Arg24, Lys27, and Arg31 of the beta-sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel-binding sites of agitoxin 2 formed a basis for site-directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel-toxin interactions.     

Primary structure and expression of a sodium channel characteristic of denervated and immature rat skeletal muscle

The alpha subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino acid protein (SkM2) that is homologous to other recently cloned sodium channels, including a tetrodotoxin (TTX)-sensitive sodium channel from rat skeletal muscle (SkM1). The SkM2 protein is no more homologous to SkM1 than to the rat brain sodium channels and differs notably from SkM1 in having a longer cytoplasmic loop joining domains 1 and 2. Steady-state mRNA levels for SkM1 and SkM2 are regulated differently during development and following denervation: the SkM2 mRNA level is highest in early development, when TTX-insensitive channels predominate, but declines rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detectable in normally innervated adult skeletal muscle but increases greater than 100-fold after denervation; rat cardiac muscle has abundant SkM2 mRNA but no detectable SkM1 message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.     

Role of the C-terminus of the high-conductance calcium-activated potassium channel in channel structure and function

The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.     

A single transmembrane site in the KCNE-encoded proteins controls the specificity of KvLQT1 channel gating

KCNEs are a family of genes encoding small integral membrane proteins whose role in governing voltage-gated potassium channel gating is emerging. Whether each member of this homologous family interacts with channel proteins in the same manner is unknown; however, it is clear that the functional effect of each KCNE on channel gating is different. The specificity of KCNE1 (minK) and KCNE3 control of activation of the potassium channel KvLQT1 maps to a triplet of amino acids within the KCNE transmembrane domain by chimera analysis. We now define the structural determinants of functional specificity within this triplet. The central amino acid of the triplet (Thr-58 of minK and Val-72 of KCNE3) is essential for the specific control of voltage-dependent channel activation characteristics of both minK and KCNE3. Using site-directed mutations that substitute minK and KCNE3 residues, we determined that a hydroxylated central amino acid is necessary for the slow sigmoidal activation produced by minK. The precise spacing of the hydroxyl group was required for minK-like activation. An aliphatic amino acid substituted at position 58 of minK is capable of reproducing KCNE3-like kinetics and voltage-independent constitutive current activation. The bulk of the central residue is another critical parameter, indicating precise positioning of this portion of the KCNE proteins within the channel complex. An intermediate phenotype produced by several smaller aliphatic-substituted mutants yields conditional voltage independence that is distinct from the voltage-dependent gating process, suggesting that KCNE3 traps the channel in a stable open state. From these results, we propose a model of KCNE-potassium channel interaction where the functional consequence depends on the precise contact at a single amino acid.     

Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore.     

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure

The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.     

Molecular dynamics study of the KcsA potassium channel

The structural, dynamical, and thermodynamic properties of a model potassium channel are studied using molecular dynamics simulations. We use the recently unveiled protein structure for the KcsA potassium channel from Streptomyces lividans. Total and free energy profiles of potassium and sodium ions reveal a considerable preference for the larger potassium ions. The selectivity of the channel arises from its ability to completely solvate the potassium ions, but not the smaller sodium ions. Self-diffusion of water within the narrow selectivity filter is found to be reduced by an order of magnitude from bulk levels, whereas the wider hydrophobic section of the pore maintains near-bulk self-diffusion. Simulations examining multiple ion configurations suggest a two-ion channel. Ion diffusion is found to be reduced to approximately (1)/(3) of bulk diffusion within the selectivity filter. The reduced ion mobility does not hinder the passage of ions, as permeation appears to be driven by Coulomb repulsion within this multiple ion channel.     

Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K(+) channel.

Three-dimensional computer modeling is used to further investigate the hypothesis forwarded in the accompanying paper of an evolutionary relationship between four related families of K(+) sympoter proteins and the superfamily of K(+) channel proteins. Atomic-scale models are developed for the transmembrane regions of one member from each of the three more distinct symporter families, i.e., a TrkH protein from Escherichia coli, a KtrB protein from Aquifex aeolicus, and a Trk1,2 protein from Schizosaccharomyces pombe. The portions of the four consecutive M1-P-M2 motifs in the symporters that can be aligned with K(+) channel sequences are modeled directly from the recently determined crystal structure of the KcsA K(+) channel from Streptomyces lividans. The remaining portions are developed using our previously accumulated theoretical modeling criteria and principles. Concurrently, the use of these criteria and principles is further supported by the now verified predictions of our previous K(+) channel modeling efforts and the degree to which they are satisfied by the known structure of the KcsA protein. Thus the observed ability of the portions of the symporter models derived from the KcsA crystal structure to also satisfy the theoretical modeling criteria provides additional support for an evolutionary link with K(+) channel proteins. Efforts to further satisfy the criteria and principles suggest that the symporter proteins from fungi and plants (i.e., Trk1,2 and HKT1) form dimeric and/or tetrameric complexes in the membrane. Furthermore, analysis of the atomic-scale models in relation to the sequence conservation within and between the protein families suggests structural details for previously proposed mechanisms for the linked symport of K(+) with Na(+) and H(+). Suggestions are also given for experiments to test these structures and hypotheses.     

A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore

The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.     

A cyclic nucleotide modulated prokaryotic K+ channel

A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive (86)Rb(+) flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K(+) channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.     

Characterization of the selectivity filter of the epithelial sodium channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed alpha, beta, and gamma. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-Val(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser(592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alphaG587C or alphaS589C were permeable to K(+), and mutation of a GSS tract (positions alpha587-alpha589) to GYG resulted in a moderately K(+)-selective channel. Our results suggest that the C-terminal portion of the pore region within the alpha-subunit contributes to the selectivity filter of ENaC.     

Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.     

Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites

Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.     

Reversible protein phosphorylation regulates the activity of the slow-vacuolar ion channel

Protein storage vacuoles (PSVs) within barley ( Hordeum vulgare ) aleurone cells contain abundant K, Ca, Mg and P reserves. These minerals are transported from the PSV and are used to support growth of the embryo. In this study, the regulation of transport through slow-vacuolar (SV) ion channels in the tonoplast of barley aleurone PSVs was examined using the patch—clamp technique. Okadaic acid (OA), an inhibitor of protein phosphatase types 1 and 2A, reduced whole-vacuole SV currents by 60%. This inhibition by OA was overcome by exogenous calcineurin. Adding ATP (200 µM) to the bath solution as a substrate for kinase activity decreased SV channel activity by 70%. This reduction in activity was prevented by the kinase inhibitor H-7. From these data, it is concluded that protein phosphorylation can inhibit SV channel activity, and that both the protein kinase and protein phosphatase involved in this regulation are present at the PSV tonoplast. Whole-vacuolar SV currents were significantly higher when 2 mM ATP was used to bathe PSVs than with 200 µM ATP. Calmodulin-like domain protein kinase (CDPK) at either ATP concentration increased SV channel activity by ∼ 150%, implying that protein phosphorylation can also stimulate SV channel activity. When PSVs were treated with the ATP analog AMP-PNP, SV channel activity was not reduced. Hence, ATP hydrolysis is not essential for sustained SV channel activity. A model in which SV channel activity is regulated by protein phosphorylation at two sites is presented.     

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel.     

Molecular model of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel.

The structure of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel from bovine retinal rod photoreceptors has been modeled by analogy to the crystal structure of the homologous cyclic AMP-binding domain of catabolite gene activator protein (CAP). The modeled cyclic GMP-binding domain has a three-residue deletion and a five-residue insertion between beta strands compared to CAP. The major interactions of the ion channel with cyclic GMP are similar to those observed for cyclic AMP bound to CAP and predicted for cGMP bound to the cGMP-dependent protein kinase: Gly 543 and Glu 544 make hydrogen-bond interactions with the ribose 2'-OH, Arg 559 forms an ion pair with the charged phosphate oxygen, and Thr 560 forms hydrogen-bond interactions with an exocyclic phosphate oxygen and with the 2-amino group of cGMP. Three additional potential interactions were predicted from the model structure. Ile 545 O and Ser 546 OH form hydrogen-bond interactions with an exocyclic phosphate oxygen, and Phe 533 may interact with the aromatic ring of cGMP. This model is in agreement with both the analogue binding experiments and the mutational analysis of Thr 560.     

Primary structure of squid sodium channel deduced from the complementary DNA sequence.

The complete amino acid sequence of a sodium channel from squid Loligo bleekeri has been deduced by cloning and sequence analysis of the complementary DNA. The deduced sequence revealed an organization virtually identical to the vertebrate sodium channel proteins; four homologous domains containing all six membrane-spanning structures are repeated in tandem with connecting linkers of various sizes. A unique feature of the squid Na channel is the 1,522 residue sequence, approximately three fourths of those of the rat sodium channels I, II and III.     

An SS1-SS2 beta-barrel structure for the voltage-activated potassium channel.

To examine the feasibility of a beta structure for the pore-lining region of the voltage-gated potassium channel, we have characterized a family of 12 antiparallel beta-barrels. Each is comprised of four identical pairs of beta-strands organized with approximate 4-fold symmetry about a channel axis. The C- and N-termini of the beta-strand pairs are assumed to be at the extracellular end of the channel, and each pair is connected by a hairpin turn at the intracellular end of the channel. The models differ in the residues located in the hairpin turn and in the orientation of the two strands of each pair in the barrel, i.e. whether the C-terminus of a pair is clockwise (CW) or counterclockwise (CCW) from the N-terminus when the channel is viewed from outside the cell. Following known structure precedents and potential energy predictions, the barrel is assumed to be right-twisting in all cases. All models have crowded layers of inward-projecting aromatic side-chains near the center of the channel which could regulate channel selectivity. The models with an odd number of amino acids in the hairpin turn have the advantage of predicting that F433 points into the barrel, but the disadvantage that V438 does not. Of these models, two of the models are most consistent with the external tetraethylammonium (TEA) block data, and of those, one (T439 CCW 3:5) is most consistent with the internal TEA block data.     

Quaternary structure of the chloride channel ClC-2

The chloride channel ClC-2 is thought to be essential for chloride homeostasis in neurons and critical for chloride secretion by the developing respiratory tract. In the present work, we investigated the quaternary structure of ClC-2 required to mediate chloride conduction. We found using chemical cross-linking and a novel PAGE system that tagged ClC-2 expressed in Sf9 cells exists as oligomers. Fusion of membranes from Sf9 cells expressing this protein confers double-barreled channel activity, with each pore exhibiting a unitary conductance of 32 pS. Polyhistidine-tagged ClC-2 from Sf9 cells can be purified as monomers, dimers, and tetramers. Purified, reconstituted ClC-2 monomers do not possess channel function whereas both purified ClC-2 dimers and tetramers do mediate chloride flux. In planar bilayers, reconstitution of dimeric ClC-2 leads to the appearance of a single, anion selective 32 pS pore, and tetrameric ClC-2 confers double-barreled channel activity similar to that observed in Sf9 membranes. These reconstitution studies suggest that a ClC-2 dimer is the minimum functional structure and that ClC-2 tetramers likely mediate double-barreled channel function.