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Calcium- and sodium- activated potassium channels are members of the 6TM family of K channels which comprises the voltage-gated KV subfamilies, including the KCNQ subfamily, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 6 or 7TM) and the Ca2+- and Na+-activated SK subfamily (nomenclature as agreed by the NC-IUPHAR Subcommittee on Calcium- and sodium-activated potassium channels [125]). As for the 2TM family, the pore-forming a subunits form tetramers and heteromeric channels may be formed within subfamilies (e.g. KV1.1 with KV1.2; KCNQ2 with KCNQ3).

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel.

Calcium- and sodium- activated potassium channels are members of the 6TM family of K channels which comprises the voltage-gated KV subfamilies, including the KCNQ subfamily, the EAG subfamily (which includes herg channels), the Ca2+-activated Slo subfamily (actually with 6 or 7TM) and the Ca2+- and Na+-activated SK subfamily (nomenclature as agreed by the NC-IUPHAR Subcommittee on Calcium- and sodium-activated potassium channels [124]). As for the 2TM family, the pore-forming a subunits form tetramers and heteromeric channels may be formed within subfamilies (e.g. KV1.1 with KV1.2; KCNQ2 with KCNQ3).

Potassium-selective channels are the largest and most diverse group of ion channels, represented by some 70 known loci in the mammalian genome. The first cloned potassium channel gene was the Drosophila voltage-gated shaker channel, and this was rapidly followed by the identification of other

This summary article presents an overview of the molecular relationships among the voltage-gated sodium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the sodium channel family can be found athttp://www.iuphar-db.org/iuphar-ic/.

Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr(250) or Val(275) in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(250) and Val(275) line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobic interactions with the pi-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside.

UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high-throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of ∼200 nM and 1 : 1 stoichiometry. A closely related compound, CP-190,325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is significantly less potent. Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at −50 mV enhances the channel's sensitivity to UK-78,282. Decreasing the number of inactivated channels by exposure to ∼160 mM external K+ decreases the sensitivity to UK-78,282. Mutations that alter the rate of C-type inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between τh and IC50 values. Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage-gated K+ channel, Kv1.4. UK-78,282 effectively suppresses human T-lymphocyte activation. British Journal of Pharmacology (1999) 126, 1707–1716; doi:10.1038/sj.bjp.0702480

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.

We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene, Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3 (Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals approximately 3-kilobase pair Kv1.7 transcripts in mouse heart and skeletal muscle. In situ hybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction.

The Kv1.3 potassium channel in T lymphocytes plays a major role in mitogen-induced activation and is widely recognized as a potential therapeutic target for immunosuppressive agents. Availability of s

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

The small-conductance calcium-activated K(+) channel SK3 (SKCa3/KCNN3) regulates electrical excitability and neurotransmitter release in monoaminergic neurons, and has been implicated in schizophrenia, ataxia and anorexia nervosa. We have identified a novel SK3 transcript, SK3-1B that utilizes an alternative first exon (exon 1B), but is otherwise identical to SK3. SK3-1B, mRNA is widely distributed in human tissues and is present at 20-60% of SK3 in the brain. The SK3-1B protein lacks the N-terminus and first transmembrane segment, and begins eight residues upstream of the second transmembrane segment. When expressed alone, SK3-1B did not produce functional channels, but selectively suppressed endogenous SK3 currents in the pheochromocytoma cell line, PC12, in a dominant-negative fashion. This dominant inhibitory effect extended to other members of the SK subfamily, but not to voltage-gated K(+) channels, and appears to be due to intracellular trapping of endogenous SK channels. The effect of SK3-1B expression is very similar to that produced by expression of the rare SK3 truncation allele, SK3-Delta, found in a patient with schizophrenia. Regulation of SK3 and SK3-1B levels may provide a potent mechanism to titrate neuronal firing rates and neurotransmitter release in monoaminergic neurons, and alterations in the relative abundance of these proteins could contribute to abnormal neuronal excitability, and to the pathogenesis of schizophrenia.

Peptidyl toxins are used extensively to determine the pharmacology of ion channels. Four families of peptides have been purified from scorpion venom. In this article, the classification of K + -channel-blocking peptides belonging to family 2 peptides and comprising 30–40 amino acids linked by three or four disulfide bridges, will be discussed. Evidence is provided for the existence of 12 molecular subfamilies, named α-KTx1–12, containing 49 different peptides. Because of the pharmacological divergence of these peptides, the principle of classification was based on a primary sequence alignment, combined with maximum parsimony and Neighbour-Joining analysis.

The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10(4) to 5 x 10(5) functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a approximately 2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of approximately 64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the approximately 64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of approximately 65 x 65 A. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.

A highly conserved motif, GYGD, contributes to the formation of the ion selectivity filter in voltage-gated K+ channels and is thought to interact with the scorpion toxin residue, Lys27. By probing the pore of the Kv1.3 channel with synthetic kaliotoxin-Lys27 mutants, each containing a non-natural lysine analog of a different length, and using mutant cycle analysis, we determined the spatial locations of Tyr400 and Asp402 in the GYGD motif, relative to His404 located at the base of the outer vestibule. Our data indicate that the terminal amines of the shorter Lys27 analogs lie close to His404 and to Asp402, while Lys27 itself interacts with Tyr400. Based on these data, we developed a molecular model of this region of the channel. The junction between the outer vestibule and the pore is defined by a ring ( approximately 8-9-A diameter) formed from alternating Asp402 and His404 residues. Tyr400 lies 4-6 A deeper into the pore, and its interaction with kaliotoxin-Lys27 is in competition with K+ ions. Studies with dimeric Kv1.3 constructs suggest that two Tyr400 residues in the tetramer are sufficient to bind K+ ions. Thus, at least part of the K+ channel signature sequence extends into a shallow trough at the center of a wide external vestibule.

In the article on Novel truncated isoform of SK3 potassium channel is a potent dominant-negative regulator of SK currents: implications in schizophrenia is described a potent dominant-negative truncated isoform of the SK3 potassium channel, SK3-1B. In the figure above, confocal fluorescence microscopy demonstrates the colocalization of functional SK3 channel protein with the inhibitory SK31B isoform. COS-7 cells that natively do not express SK3 were transfected with both constructs, SK3-1B being tagged with green fluorescent protein (GFP). Top panel: Cells stained with antibody (red) specific for only the full-length SK3 channel protein. Middle panel: GFP fluorescence (green) showing the distribution of SK3-1B protein. Lower panel: Overlay of the two images shows their colocalization, seen as yellow. (N indicates the nucleus). In the article, electrophysiology and confocal microscopy show that this colocalization is the mechanism of dominant-negative inhibition of channel function, and the potential implications of this mechanism in the pathophysiology of schizophrenia are discussed. For more information on this topic, please see the article by Tomita et al 524–535 of this issue. Molecular Psychiatry (2003) 8, 460 & 2003 Nature Publishing Group All rights reserved 1359-4184/03 $25.00

The IgG subclass and the IgM isotype response to immunization with particulate bovine tubular basement membrane (TBM) and adjuvants was studied in Brown-Norway rats receiving daily injections of a stable analogue of PGE, (M-PGE,). M-PGE, slightly reduced the average quantity of circulating TBM antibody as well as the average quantity of eluted IgG per gram of renal tissue as compared to controls. However, M-PGEI did not qualitatively affect the distribution of the IgG subclass or IgM isotype response to TBM. The IgG response, which occurred predominantly in the IgG, and IgG,, subclasses, increased from Days 8 to 14 after immunization, while the IgM response decreased over the same time period. The percentage of TBM antibody in the IgGZb subclass was markedly decreased as compared to the percentage of IgG2b antibody in total IgG. A substantial heterogeneity in the IgG subclass response was noted among individual rats with IgGl constituting from 46 to 82% of circulating TBM antibody. Although no correlation between the IgG subclass response and the severity of tubulointerstitial nephritis was noted, het- erogeneity in the IgG subclass response to autoantigens may, nevertheless, theoretically play an important role in the pathogenesis of autoimmune inflammatory phenomena. o 1987 society for Experimental Biology and Medicine.

We have cloned DNA segments containing the J κ genes from LOUVAIN rat liver, and have determined their nucleotide sequence. Seven readily identifiable J κ -coding regions (six expressible) are evident in the rat, compared with five in the mouse (four expressible). The two additional J segments in the rat appear to be the result of two sequential gene duplications occurring since the divergence of rats and mice. The first involved a homologous but unequal crossing-over in a 14 bp region spanning the 3′ end of the coding region of J1 and J2. The second involved a crossing-over following unequal pairing of the two newly duplicated regions. We propose that the probability of a second duplication was greatly increased following the first as a result of the increased target for unequal pairing (370 bp of good homology versus 27 bp in the original pairing). Comparisons of rat and mouse J genes show a surprisingly high degree of sequence conservation, both inside and outside the coding regions, similar to the pattern we reported previously for the kappa constant-region gene. This provides additional evidence that constraints exist on the nucleotide sequences of these genes independent of the function of the encoded proteins.

Slipped-strand mispairing (SSM) may play an major role in repetitive DNA sequence evolution by generating large numbers of short frameshift mutations within simple tandem repeats. Here we examine the frequency and size spectrum of frameshifts generated within poly-CA/TG sequences inserted into bacteriophage M13 in Escherichia coli hosts. The frequency of detectable frameshifts within a 40 bp tract of poly-CA/TG is greater than one percent and increases more than linearly with length, being lower by a factor of four in a 22 bp target sequence. The frequency increases more than 13-fold in mutL and mutS host cells, suggesting that a high proportion of frameshift events are normally repaired by methyl-directed mismatch repair. Of the 87 sequenced frameshifts in this study, 96% result from deletion or insertion of only or two 2 bp repeat units. The most frequent events are 2 bp deletions, 2 bp insertions, and 4 bp deletions, the relative frequencies of these events being about 18:6:1.

We have analyzed protein-coding sequences of Escherichia coli and find that codon-pair utilization is highly biased, reflecting overrepresentation or underrepresentation of many pairs compared with their random expectations. This effect is over and above that contributed by nonrandomness in the use of amino acid pairs, which itself is highly evident; it is much weaker when nonadjacent codon pairs are examined and virtually disappears when pairs separated by two or three intervening codons are evaluated. There appears to be a high degree of directionality in this bias: any codon that participates in many nonrandom pairs tends to make both over- and underrepresented pairs, but preferentially as a left- or right-hand member. We show a relationship between codon-pair utilization patterns and levels of gene expression: genes encoding proteins expressed at high levels tend to contain more abundant, but more highly underrepresented, codon pairs, relative to genes expressed at low levels. The nonrandom utilization of codon pairs may be a consequence of their effects on translational efficiency, which in turn may be related to the compatibility of adjacent aminoacyl-tRNA isoacceptors at the A and P sites of a translating ribosome.

Monoclonal antibodies directed against rat kappa light chains have been generated by immunizing SJL/J mice with soluble rat immunoglobulin, followed by fusion of immune spleen cells with the P3-X63-Ag8 myeloma cell line. Monoclonal antibodies from three of these hybridoma cell lines, MAR 18.5, 80.2, and 103.6, have been extensively characterized. MAR 18.5, 80.2, and 103.6 antibodies are of the gamma 2a kappa isotype, and bind strongly to protein A, allowing easy purification. Monoclonal antibody from clone 18.5 binds equally well to Ig of both RI-1a and RI-1b allotypes, whereas 80.2 and 103.6 antibodies selectively bind to RI-1b. These monoclonal antibodies can be FITC conjugated for use as a second antibody in indirect immunofluorescence assays, or radiolabeled for use in radio immunoassays requiring a specific antirat kappa antibody. The antiallotype specific monoclonal antibodies also may be of use in the study of rat immunoglobulin genetics.

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing-over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.

We have cloned and sequenced the C-kappa (Ck) genes from seven species and subspecies of rats which have diverged over the past few million years in Australia. Comparisons of these sequences with each other and the Ck genes of the laboratory rat, Rattus norvegicus, indicate noncoding regions have accumulated fewer mutations than adjacent coding sequences, and amino acid replacing nucleotide substitutions in the coding regions have accumulated at a rate at least as great as silent changes. Exactly the opposite of both of these findings is observed when comparisons are made between Ck or other genes from more distantly related species, indicating that these features may be characteristic of Ck short-term evolutionary gene divergence. Changes in the coding regions of these genes result in a non-random distribution of amino acid substitutions on the three-dimensional alpha-carbon backbone of the Ck domain in the most serologically distinct forms of Ck. While phylogenetic relationships inferred from the Ck nucleotide sequences are in general agreement with those derived from other data, considerable differences are seen in rates of accumulation of Ck gene nucleotide substitutions vs rates of accumulation of enzyme polymorphisms.

The gamma-emitting amino acid analog, [75Se]selenomethionine, has been used as a biosynthetic label for immunoglobulins secreted by plasmacytomas in tissue culture. The secreted products are structurally intact with respect to their antibody combining sites and their class and allotype antigenic specificities. A component of [75Se]selenomethionine preparations was found to bind to fetal calf serum proteins, in a manner releasable by mercaptoethanol, but not by sodium dodecyl sulfate (SDS) and urea. Methods for circumventing the problems caused by this binding are described.

Parasites have coexisted with their human hosts for thousands of years and are known mainly for their harmful disease-causing role in humans. However, probiotic worm therapy is beneficial in human autoimmune diseases, suggesting the existence of immunomodulators in parasitic worms. By screening a cDNA library and searching genome databases we identified a family of peptides in parasitic worms that share sequence similarity and evolutionary relatedness to potassium channel-blocking peptides secreted by venomous sea anemones. AcK1, a 51-residue secreted peptide of the hookworm Ancylostoma caninum, and BmK1, the C-terminal domain of a 413-residue zinc metalloprotease from the filarial worm Brugia malayi, share structural similarity to ShK and BgK peptides from sea anemones. These peptides block cloned and native human T-cell Kv1.3 channels at nanomolar to low micromolar concentrations. BmK2, an analog of BmK1, blocks Kv1.3 with an IC50 of 2 nM and exhibits >4000-fold selectivity for Kv1.3 over Kv1.1, Kv1.2, Kv1.6, Kv3.2, KCa3.1, K2P3.1. These peptides suppress proliferation of effector memory T cells that use Kv1.3 channels to regulate membrane potential, without affecting other T cell subsets that are not dependent on Kv1.3. They inhibit cytokine production and suppress the in vivo delayed type hypersensitivity response. Our results provide a mechanistic basis for pro-biotic worm therapy in human autoimmune disease, and suggest that these or related peptides and proteins could supplant the need for worm therapy.