Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity

K+ channels are known through electrophysiology and pharmacology to be an exceptionally diverse group of channels. Molecular studies of the Shaker (Sh) locus in Drosophila have provided the first glimpse of K+ channel structure. The sequences of several Sh cDNA clones have been reported; none are identical. We have isolated and examined 18 additional Sh cDNAs in an attempt to understand the origin, extent, and significance of the variability. The diversity is extensive: we have already identified cDNAs representing at least nine distinct types, and Sh could potentially encode 24 or more products. This diversity, however, fits a simple pattern in which variable 3' and 5' ends are spliced onto a central constant region to yield different cDNA types. These different Sh cDNAs encode proteins with distinct structural features.     

Molecular cloning and functional expression of a potassium channel cDNA isolated from a rat cardiac library

A full-length K+ channel cDNA (RHK1) was isolated from a rat cardiac library using the polymerase chain reaction (PCR) method and degenerate oligonucleotide primers derived from K+ channel sequences conserved between Drosophila Shaker H4 and mouse brain MBK1. Although RHK1 was isolated from heart, its expression was found in both heart and brain. The RHK1-encoded protein, when expressed in Xenopus oocytes, gated a 4-aminopyridine (4-AP)-sensitive transient outward current. This current is similar to the transient outward current measured in rat ventricular myocytes with respect to voltage-dependence of activation and inactivation, time course of activation and inactivation, and pharmacology.     

Molecular diversity and functional evolution of scorpion potassium channel toxins

Scorpion toxins affecting K(+) channels (KTxs) represent important pharmacological tools and potential drug candidates. Here, we report molecular characterization of seven new KTxs in the scorpion Mesobuthus eupeus by cDNA cloning combined with biochemical approaches. Comparative modeling supports that all these KTxs share a conserved cysteine-stabilized α-helix/β-sheet structural motif despite the differences in protein sequence and size. We investigated functional diversification of two orthologous α-KTxs (MeuTXKα1 from M. eupeus and BmP01 from Mesobuthus martensii) by comparing their K(+) channel-blocking activities. Pharmacologically, MeuTXKα1 selectively blocked Kv1.3 channel with nanomolar affinity (IC(50), 2.36 ± 0.9 nM), whereas only 35% of Kv1.1 currents were inhibited at 3 μM concentration, showing more than 1271-fold selectivity for Kv1.3 over Kv1.1. This peptide displayed a weak effect on Drosophila Shaker channel and no activity on Kv1.2, Kv1.4, Kv1.5, Kv1.6, and human ether-a-go-go-related gene (hERG) K(+) channels. Although BmB01 and MeuTXKα1 have a similar channel spectrum, their affinity and selectivity for these channels largely varies. In comparison with MeuTXKα1, BmP01 only exhibits a submicromolar affinity (IC(50), 133.72 ± 10.98 nM) for Kv1.3, showing 57-fold less activity than MeuTXKα1. Moreover, it lacks the ability to distinguish between Kv1.1 and Kv1.3. We also found that MeuTXKα1 inhibited the proliferation of activated T cells induced by phorbol myristate acetate and ionomycin at micromolar concentrations. Our results demonstrate that accelerated evolution drives affinity variations of orthologous α-KTxs on Kv channels and indicate that MeuTXKα1 is a promising candidate to develop an immune modulation agent for human autoimmune diseases.     

Molecular cloning and functional expression of the human sodium channel beta1B subunit, a novel splicing variant of the beta1 subunit.

The voltage gated sodium channel comprises a pore-forming alpha subunit and regulatory beta subunits. We report here the identification and characterization of a novel splicing variant of the human beta1 subunit, termed beta1B. The 807 bp open reading frame of the human beta1Beta subunit encodes a 268 residue protein with a calculated molecular mass of 30.4 kDa. The novel human beta1B subunit shares an identical N-terminal half (residues 1-149) with the human beta1 subunit, but contains a novel C-terminal half (residues 150-268) of less than 17% sequence identity with the human beta1 subunit. The C-terminal region of the human beta1B is also significantly different from that of the rat beta1A subunit, sharing less than 33% sequence identity. Tissue distribution studies reveal that the human beta1Beta subunit is expressed predominantly in human brain, spinal cord, dorsal root ganglion and skeletal muscle. Functional studies in oocytes demonstrate that the human beta1B subunit increases the ionic current when coexpressed with the tetrodotoxin sensitive channel, NaV1.2, without significantly changing voltage dependent kinetics and steady-state properties, thus distinguishing it from the human beta1 and rat beta1A subunits.     

Molecular cloning and functional analysis of a voltage-gated potassium channel in lymphocytes from sea perch, Lateolabrax japonicus

Voltage-gated potassium (Kv) channels on cell plasma membrane play an important role in both excitable cells and non-excitable cells and Kv1 subfamily is most extensively studied channel in mammalian cells. Recently, this potassium channel was reported to control processes inside mammalian T lymphocytes such as cell proliferation and volume regulation. Little is known about Kv1 channels in fish. We have postulated the presence of such a channel in lymphocytes and speculated its potential role in immunoregulation in ?sh. Employing speci?c primers and RNA template, we cloned a segment of a novel gene from sea perch blood sample and subsequently obtained a full cDNA sequence using RACE approach. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel Kv channel gene, designated as spKv1.3, which exhibits homologous domains to the members of Kv1.3 family, but it differs notably from some other members of that family at the carboxyl terminus. Full-length of spKv1.3 cDNA is 2152 bp with a 1440 bp open reading frame encoding a protein of 480 amino acids. SpKv1.3 gene is expressed in all of the tested organs and tissues of sea perch. To assess the postulated immune function of spKv1.3, we stimulated lymphocytes with LPS and/or channel blocker 4-AP. Expression levels of messenger RNA (mRNA) of spKv1.3 under stimulation conditions were measured by quantitative RT-PCR. The results showed that LPS can motivate the up-regulation of spKv1.3 expression significantly. Interestingly, we found for the first time that 4-AP with LPS can also increase the spKv1.3 mRNA expression levels in time course. Although 4-AP could block potassium channels physically, we speculated that its effect on blockage of potassium channel may start up an alternative mechanism which feed back and evoke the spKv1.3 mRNA expression.     

KCNQ5, a novel potassium channel broadly expressed in brain, mediates M-type currents

KCNQ2 and KCNQ3, both of which are mutated in a type of human neonatal epilepsy, form heteromeric potassium channels that are expressed in broad regions of the brain. The associated current may be identical to the M-current, an important regulator of neuronal excitability. We now show that the RNA encoding the novel KCNQ5 channel is also expressed in brain and in sympathetic ganglia where it overlaps largely with KCNQ2 and KCNQ3. In addition, it is expressed in skeletal muscle. KCNQ5 yields currents that activate slowly with depolarization and can form heteromeric channels with KCNQ3. Currents expressed from KCNQ5 have voltage dependences and inhibitor sensitivities in common with M-currents. They are also inhibited by M1 muscarinic receptor activation. A KCNQ5 splice variant found in skeletal muscle displays altered gating kinetics. This indicates a molecular diversity of channels yielding M-type currents and suggests a role for KCNQ5 in the regulation of neuronal excitability.     

Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full-length cDNA for Kv2-catfish, a novel K(+) channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56-59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage-gated K(+) channels. The Kv2-catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in XENOPUS: oocytes were selective for K(+), and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two-electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mM. The channels showed voltage-dependent activation, and did not inactivate within 200 ms. Functionally, Kv2-catfish is a voltage-gated, delayed rectifier K(+) channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K(+) channels, being related equally well to Kv2.1 and Kv2.2.     

Molecular cloning, characterization, and genomic localization of a human potassium channel gene.

Potassium (K+) channels are critical for a variety of cell functions, including modulation of action potentials, determination of resting membrane potential, and development of memory and learning. In addition to their role in regulating myocyte excitability, cardiac K+ channels control heart rate and coronary vascular tone and are implicated in the development of arrhythmias. We report here the cloning and sequencing of a k+ channel gene, KCNA1, derived from a human cardiac cDNA library and the chromosomal localization of the corresponding genomic clone. Oligonucleotides based on a delayed rectifier K+ channel gene were used in PCR reactions with human genomic DNA to amplify the S4-S6 regions of several different K+ channel genes. These sequences were used to isolate clones from a human cardiac cDNA library. We sequenced one of these clones, HCK1. HCK1 contains putative S2-S6 domains and shares approximately 70% sequence homology with previously isolated Shaker homologues. HCK1 was used to screen human cosmid libraries and a genomic clone was isolated. By sequencing the genomic clones, a putative S1 domain and translation initiation sequences were identified. Genomic mapping using human-rodent somatic cell panels and in situ hybridization with human metaphase chromosomes have localized KCNA1 to the distal short arm of human chromosome 12. This work is an important step in the study of human cardiac K+ channel structure and function and will be of use in the study of human inherited disease.     

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.     

Molecular cloning and characterization of LKv1, a novel voltage-gated potassium channel in leech

We have cloned a novel voltage-gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage-gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I(K). Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage-gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker-related K channels in jellyfish. Thus, this analysis indicates that this group of voltage-gated K channels contains several evolutionarily divergent lineages.     

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT(1Pem) receptor protein of 591 amino acids. Transient expression of 5-HT(1Pem) in HEK293 cells demonstrated a saturable [3H]-5-HT binding with a Kd of 10.43+/-1.13 nM and Bmax of 1.53+/-0.06 pmol/mg. The putative 5-HT(1Pem) receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT(1Pem) receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.     

Molecular cloning and functional expression of a Drosophila corazonin receptor

Here we report a novel method for selecting human antibody fragments from nonimmunized variable domain libraries. The antibody fragments are selected on the basis of stabilization of the variable domain fragment (F(v)) in the presence of target antigens ("open sandwich selection"). One variable domain is displayed on phages and another is prepared as soluble molecules. These two reagents are mixed with the biotinylated target molecule and ternary complexes are captured by using streptavidin-conjugated magnet beads. After extensive washing, enriched clones are eluted by using target antigen. Some of the clones selected after 3 rounds are prepared as soluble domains, which then undergo another selection process. We obtained several human antibody fragments specific for human soluble erythropoietin receptor by using this method. Our method minimizes several of the disadvantages associated with human antibody selection through a phage-display system, such as construction of a large-scale library, deletion of genes during selection, and nonspecific binding.     

Structural and functional diversity of acidic scorpion potassium channel toxins

Background

Although the basic scorpion K⁺ channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K⁺ channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly.

Principal Findings

Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif that has no apparent α-helixs and β-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a K_d of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-β-sheet (CS-α/β) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel.

Conclusions/Significance

These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.     

Molecular features of an alcohol binding site in a neuronal potassium channel

Aliphatic alcohols (1-alkanols) selectively inhibit the neuronal Shaw2 K(+) channel at an internal binding site. This inhibition is conferred by a sequence of 13 residues that constitutes the S4-S5 loop in the pore-forming subunit. Here, we combined functional and structural approaches to gain insights into the molecular basis of this interaction. To infer the forces that are involved, we employed a fast concentration-clamp method (10-90% exchange time = 800 micros) to examine the kinetics of the interaction of three members of the homologous series of 1-alkanols (ethanol, 1-butanol, and 1-hexanol) with Shaw2 K(+) channels in Xenopus oocyte inside-out patches. As expected for a second-order mechanism involving a receptor site, only the observed association rate constants were linearly dependent on the 1-alkanol concentration. While the alkyl chain length modestly influenced the dissociation rate constants (decreasing only approximately 2-fold between ethanol and 1-hexanol), the second-order association rate constants increased e-fold per carbon atom. Thus, hydrophobic interactions govern the probability of productive collisions at the 1-alkanol binding site, and short-range polar interactions help to stabilize the complex. We also examined the relationship between the energetics of 1-alkanol binding and the structural properties of the S4-S5 loop. Circular dichroism spectroscopy applied to peptides corresponding to the S4-S5 loop of various K(+) channels revealed a correlation between the apparent binding affinity of the 1-alkanol binding site and the alpha-helical propensity of the S4-S5 loop. The data suggest that amphiphilic interactions at the Shaw2 1-alkanol binding site depend on specific structural constraints in the pore-forming subunit of the channel.     

Molecular cloning of a pore-forming subunit (Kir6.2 gene) of the ATP-sensitive potassium channel in the bullfrog,Rana catesbeiana Shaw

A cDNA sequence encoding a pore-forming subunit of ATP-sensitive potassium channel (Kir6.2 gene) of the bullfrog, Rana catesbeiana Shaw, termed RcKir6.2, was isolated from a liver cDNA library. The cDNA contained a single open reading frame of 1,173 bp encoding 391 amino acids with a calculated molecular mass of 42.9 kDa, which has a structural motif (a GFG motif) of the putative pore-forming loop of Kir6.2. Analysis of its phlyogenetic position revealed that the RcKir6.2 is close to Kir6.2 of rabbits. The predicted amino acid sequence shared sequence identity with Kir6.2 of Homo sapiens, Cavia porcellus, Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus by 95.9, 95.6, 96.7, 96.7 and 99.7%, respectively. Expression of RcKir6.2 was detected in various tissues, including heart, kidney, liver, lung, spleen, and stomach of the bullfrog.     

Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K(i) values of 1.18 x 10(-10) M and 9.53 x 10(-11) M, respectively. The recombinant cystatins were found to be thermally stable up to 60 degrees C. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing.     

Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni

A cDNA encoding a high-affinity Na(+)-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na(+)-dependent [(3)H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [(3)H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [(3)H]-choline in the absence of Na(+). The Na(+)-dependent component of [(3)H]-choline uptake by TrnCHT-infected cells was saturable with a K(m) for choline transport of 8.4 microM. Several compounds reported to be potent blockers of [(3)H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (K(i)=4.1 microM) and two oxoquinuclidium analogues of choline, quireston-A (K(i) approximately 10 microM) and quireston (K(i) approximately 100 microM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na(+)-independent uptake of [(3)H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.     

Molecular cloning and functional expression of Ca(v)3.1c, a T-type calcium channel from human brain

Low voltage-activated T-type calcium channels are encoded by a family of at least three genes, with additional diversity created by alternative splicing. This study describes the cloning of the human brain alpha1G, which is a novel isoform, Ca(v)3.1c. Comparison of this sequence to genomic sequences deposited in the GenBank allowed us to identify the intron/exon boundaries of the human CACNA1G gene. A full-length cDNA was constructed, then used to generate a stably-transfected mammalian cell line. The resulting currents were analyzed for their voltage- and time-dependent properties. These properties identify this gene as encoding a T-type Ca(2+) channel.     

Molecular cloning,expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.