Genomic organization and chromosomal localization of the murine 2 P domain potassium channel gene Kcnk8: conservation of gene structure in 2 P domain potassium channels

A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.     

双孔钾通道TREK-1 结构、分布、调控因素及其抗癫痫作用的研究进展

：钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70 余种钾离子通道亚型,其中双孔钾离 子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统 的单孔钾离子通道差异很大。双孔钾离子通道,具有4 个跨膜片段,形成独特的2 个孔道结构域,主要介导背景钾电流。由于其介 导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1 几乎表达于机 体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生 理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

双孔钾通道TREK-1结构、分布、调控因素及其抗癫痫作用的研究进展

钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70余种钾离子通道亚型,其中双孔钾离子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统的单孔钾离子通道差异很大。双孔钾离子通道,具有4个跨膜片段,形成独特的2个孔道结构域,主要介导背景钾电流。由于其介导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1几乎表达于机体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

A central role for the T1 domain in voltage-gated potassium channel formation and function

To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the full-length functional channel.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.     

The role of the N terminus and transmembrane domain of TRPM8 in channel localization and tetramerization

Transient receptor potential (TRP) channels are a family of cation channels involved in diverse cellular functions. They are composed of a transmembrane domain of six putative transmembrane segments flanked by large N- and C-terminal cytoplasmic domains. The melastatin subfamily (TRPM) channels have N-terminal domains of approximately 700 amino acids with four regions of shared homology and C-terminal domains containing the conserved TRP domain followed by a coiled-coil region. Here we investigated the effects of N- and C-terminal deletions on the cold and menthol receptor, TRPM8, expressed heterologously in Sf21 insect cells. Patch-clamp electrophysiology was used to study channel activity and revealed that only deletion of the first 39 amino acids was tolerated by the channel. Further N-terminal truncation or any C-terminal deletions prevented proper TRPM8 function. Confocal microscopy with immunofluorescence revealed that amino acids 40-86 are required for localization to the plasma membrane. Furthermore, analysis of deletion mutant oligomerization shows that the transmembrane domain is sufficient for TPRM8 assembly into tetramers. TRPM8 channels with C-terminal deletions tetramerize and localize properly but are inactive, indicating that although not essential for tetramerization and localization, the C terminus is critical for proper function of the channel sensor and/or gate.     

A voltage-sensor water pore

Voltage-sensor (VS) domains cause the pore of voltage-gated ion channels to open and close in response to changes in transmembrane potential. Recent experimental studies suggest that VS domains are independent structural units. This independence is revealed dramatically by a voltage-dependent proton-selective channel (Hv), which has a sequence homologous to the VS domains of voltage-gated potassium channels (Kv). Here we show by means of molecular dynamics simulations that the isolated open-state VS domain of the KvAP channel in a lipid membrane has a configuration consistent with a water channel, which we propose as a common feature underlying the conductance of protons, and perhaps other cations, through VS domains.     

Tetramerization of the AKT1 plant potassium channel involves its C-terminal cytoplasmic domain.

All plant channels identified so far show high conservation throughout the polypeptide sequence except in the ankyrin domain which is present only in those closely related to AKT1. In this study, the architecture of the AKT1 protein has been investigated. AKT1 polypeptides expressed in the baculovirus/Sf9 cells system were found to assemble into tetramers as observed with animal Shaker-like potassium channel subunits. The AKT1 C-terminal intracytoplasmic region (downstream from the transmembrane domain) alone formed tetrameric structures when expressed in Sf9 cells, revealing a tetramerization process different from that of Shaker channels. Tests of subfragments from this sequence in the two-hybrid system detected two kinds of interaction. The first, involving two identical segments (amino acids 371-516), would form a contact between subunits, probably via their putative cyclic nucleotide-binding domains. The second interaction was found between the last 81 amino acids of the protein and a region lying between the channel hydrophobic core and the putative cyclic nucleotide-binding domain. As the interacting regions are highly conserved in all known plant potassium channels, the structural organization of AKT1 is likely to extend to these channels. The significance of this model with respect to animal cyclic nucleotide-gated channels is also discussed.     

Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution

BACKGROUND: The voltage-gated potassium channel Shaker from Drosophila consists of a tetramer of identical subunits, each containing six transmembrane segments. The atomic structure of a bacterial homolog, the potassium channel KcsA, is much smaller than Shaker. It does not have a voltage sensor and other important domains like the N-terminal tetramerization (T1) domain. The structure of these additional elements has to be studied in the more complex voltage-gated channels. RESULTS: We determined the three-dimensional structure of the entire Shaker channel at 2.5 nm resolution using electron microscopy. The four-fold symmetric structure shows a large and a small domain linked by thin 2 nm long connectors. To interpret the structure, we used the crystal structures of the isolated T1 domain and the KcsA channel. A unique density assignment was made based on the symmetry and dimensions of the crystal structures and domains, identifying the smaller domain as the cytoplasmic mass of Shaker containing T1 and the larger domain as embedded in the membrane. CONCLUSIONS: The two-domain architecture of the Shaker channel is consistent with the recently proposed "hanging gondola" model for the T1 domain, putting the T1 domain at a distance from the membrane domain but attached to it by thin connectors. The space between the two domains is sufficient to permit cytoplasmic access of ions and the N-terminal inactivation domain to the pore region. A hanging gondola architecture has also been observed in the nicotinic acetylcholine receptor and the KcsA structure, suggesting that it is a common element of ion channels.     

Homology models of the tetramerization domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6

The homology models of the tetramerization (T1) domain of six eukaryotic potassium channels, Kv1.1-Kv1.6, were constructed based on the crystal structure of the Shaker T1 domain. The results of amino acid sequence alignment indicate that the T1 domains of these K+ channels are highly conserved, with the similarities varying from 77% between Shaker and Kv1.6 to 93% between Kv1.2 and Kv1.3. The homology models reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K+ channel. These models also show that each T1 monomer consists of three distinct layers, with N-terminal layer 1 and C-terminal layer 3 facing the cytoplasm and the membrane, respectively. Layer 2 exhibits the highest structural conservation because it is located around the central hydrophobic core. For each Kv channel, four identical subunits assemble into the homotetramer architecture around a four-fold axis through the hydrogen bonds and salt bridges formed by 15 highly conserved polar residues. The narrowest opening of the pore is formed by the four conserved residues corresponding to R115 of the Shaker T1 domain. The homology models of these Kv T1 domains provide particularly attractive targets for further structure-based studies.     

Crystal structure of the cytoplasmic domain of the chloride channel ClC-0

Ion channels are frequently organized in a modular fashion and consist of a membrane-embedded pore domain and a soluble regulatory domain. A similar organization is found for the ClC family of Cl- channels and transporters. Here, we describe the crystal structure of the cytoplasmic domain of ClC-0, the voltage-dependent Cl- channel from T. marmorata. The structure contains a folded core of two tightly interacting cystathionine beta-synthetase (CBS) subdomains. The two subdomains are connected by a 96 residue mobile linker that is disordered in the crystals. As revealed by analytical ultracentrifugation, the domains form dimers, thereby most likely extending the 2-fold symmetry of the transmembrane pore. The structure provides insight into the organization of the cytoplasmic domains within the ClC family and establishes a framework for guiding future investigations on regulatory mechanisms.     

A novel two-pore domain K+ channel,TRESK, is localized in the spinal cord

To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.     

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.     

Role of the KcsA channel cytoplasmic domain in pH-dependent gating

The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.     

Conformational dynamics of the ligand-binding domain of inward rectifier K channels as revealed by molecular dynamics simulations: toward an understanding of Kir channel gating

Inward rectifier (Kir) potassium channels are characterized by two transmembrane helices per subunit, plus an intracellular C-terminal domain that controls channel gating in response to changes in concentration of various ligands. Based on the crystal structure of the tetrameric C-terminal domain of Kir3.1, it is possible to build a homology model of the ATP-binding C-terminal domain of Kir6.2. Molecular dynamics simulations have been used to probe the dynamics of Kir C-terminal domains and to explore the relationship between their dynamics and possible mechanisms of channel gating. Multiple simulations, each of 10 ns duration, have been performed for Kir3.1 (crystal structure) and Kir6.2 (homology model), in both their monomeric and tetrameric forms. The Kir6.2 simulations were performed with and without bound ATP. The results of the simulations reveal comparable conformational stability for the crystal structure and the homology model. There is some decrease in conformational flexibility when comparing the monomers with the tetramers, corresponding mainly to the subunit interfaces in the tetramer. The beta-phosphate of ATP interacts with the side chain of K185 in the Kir6.2 model and simulations. The flexibility of the Kir6.2 tetramer is not changed greatly by the presence of bound ATP, other than in two loop regions. Principal components analysis of the simulated dynamics suggests loss of symmetry in both the Kir3.1 and Kir6.2 tetramers, consistent with "dimer-of-dimers" motion of subunits in C-terminal domains of the corresponding Kir channels. This is suggestive of a gating model in which a transition between exact tetrameric symmetry and dimer-of-dimers symmetry is associated with a change in transmembrane helix packing coupled to gating of the channel. Dimer-of-dimers motion of the C-terminal domain tetramer is also supported by coarse-grained (anisotropic network model) calculations. It is of interest that loss of exact rotational symmetry has also been suggested to play a role in gating in the bacterial Kir homolog, KirBac1.1, and in the nicotinic acetylcholine receptor channel.     

Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag-like K(+)) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization-activated cyclic nucleotide-gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the "self-liganded" conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.     

Base of the thumb domain modulates epithelial sodium channel gating

The activity of the epithelial sodium channel (ENaC) is modulated by multiple external factors, including proteases, cations, anions and shear stress. The resolved crystal structure of acid-sensing ion channel 1 (ASIC1), a structurally related ion channel, and mutagenesis studies suggest that the large extracellular region is involved in recognizing external signals that regulate channel gating. The thumb domain in the extracellular region of ASIC1 has a cylinder-like structure with a loop at its base that is in proximity to the tract connecting the extracellular region to the transmembrane domains. This loop has been proposed to have a role in transmitting proton-induced conformational changes within the extracellular region to the gate. We examined whether loops at the base of the thumb domains within ENaC subunits have a similar role in transmitting conformational changes induced by external Na(+) and shear stress. Mutations at selected sites within this loop in each of the subunits altered channel responses to both external Na(+) and shear stress. The most robust changes were observed at the site adjacent to a conserved Tyr residue. In the context of channels that have a low open probability due to retention of an inhibitory tract, mutations in the loop activated channels in a subunit-specific manner. Our data suggest that this loop has a role in modulating channel gating in response to external stimuli, and are consistent with the hypothesis that external signals trigger movements within the extracellular regions of ENaC subunits that are transmitted to the channel gate.     

N-terminal transmembrane domain of the SUR controls trafficking and gating of Kir6 channel subunits

The sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, assembles with a potassium channel subunit (Kir6) to form the ATP-sensitive potassium channel (K(ATP)) complex. Although SUR is an important regulator of Kir6, the specific SUR domain that associates with Kir6 is still unknown. All functional ABC proteins contain two transmembrane domains but some, including SUR and MRP1 (multidrug resistance protein 1), contain an extra N-terminal transmembrane domain called TMD0. The functions of any TMD0s are largely unclear. Using Xenopus oocytes to coexpress truncated SUR constructs with Kir6, we demonstrated by immunoprecipitation, single-oocyte chemiluminescence and electrophysiological measurements that the TMD0 of SUR1 strongly associated with Kir6.2 and modulated its trafficking and gating. Two TMD0 mutations, A116P and V187D, previously correlated with persistent hyperinsulinemic hypoglycemia of infancy, were found to disrupt the association between TMD0 and Kir6.2. These results underscore the importance of TMD0 in K(ATP) channel function, explaining how specific mutations within this domain result in disease, and suggest how an ABC protein has evolved to regulate a potassium channel.     

Splitting the two pore domains from TOK1 results in two cationic channels with novel functional properties.

Potassium channels are membrane-spanning proteins with several transmembrane segments and a single pore region where ion conduction takes place (Biggin, P. C., Roosild, T., and Choe, S. (2000) Curr. Opin. Struct. Biol. 4, 456-461; Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77). TOK1, a potassium channel identified in the yeast Saccharomyces cerevisiae, was the first described member from a growing new family of potassium channels with two pore domains in tandem (2P) (Ketchum, K. A., Joiner, W. J., Sellers, A. J., Kaczmarek, L. K., and Goldstein, S. A. (1995) Nature 376, 690-695). In an attempt to understand the relative contribution of each one of the 2P from TOK1 to the functional properties of this channel, we split and expressed the pore domains separately or in combination. Expression of the two domains separately rescued a potassium transport-deficient yeast mutant, suggesting that each domain forms functional potassium-permeable channels in yeast. In Xenopus laevis oocytes expression of each pore domain resulted in the appearance of unique inwardly rectifying cationic channels with novel gating and pharmacological properties. Both pore domains were poorly selective to potassium; however, upon co-expression they partially restored TOK1 channel selectivity. The single channel conductance was different in both pore domains with 7 +/- 1 (n = 12) and 15 +/- 2 (n = 12) picosiemens for the first and second domain, respectively. In light of the known structure of the Streptomyces lividans KcsA potassium channel pore (see Doyle et al. above), these results suggest a novel non-four-fold-symmetric architecture for 2P potassium-selective channels.