In mammalian cardiac cells, a variety of transient or sustained K+ currents contribute to the repolarization of action potentials. There are two main components of the delayed-rectifier sustained K+ current, I(Kr) (rapid) and I(Ks), (slow). I(Kr) is the product of the gene HERG, which is altered in the long-QT syndrome, LQT2. A channel with properties similar to those of the I(Ks) channel is produced when the cardiac protein IsK is expressed in Xenopus oocytes. However, it is a small protein with a very unusual structure for a cation channel. The LQT1 gene is another gene associated with the LQT syndrome, a disorder that causes sudden death from ventricular arrhythmias. Here we report the cloning of the full-length mouse K(V)LQT1 complementary DNA and show that K(V)LQT1 associates with IsK to form the channel underlying the I(Ks) cardiac current, which is a target of class-III anti-arrhythmic drugs and is involved in the LQT1 syndrome.

A new human weakly inward rectifying K+ channel, TWIK‐1, has been isolated. This channel is 336 amino acids long and has four transmembrane domains. Unlike other mammalian K+ channels, it contains two pore‐forming regions called P domains. Genes encoding structural homologues are present in the genome of Caenorhabditis elegans. TWIK‐1 currents expressed in Xenopus oocytes are time‐independent and present a nearly linear I‐V relationship that saturated for depolarizations positive to O mV in the presence of internal Mg2+. This inward rectification is abolished in the absence of internal Mg2+. TWIK‐1 has a unitary conductance of 34 pS and a kinetic behaviour that is dependent on the membrane potential. In the presence of internal Mg2+, the mean open times are 0.3 and 1.9 ms at −80 and +80 mV, respectively. The channel activity is up‐regulated by activation of protein kinase C and down‐regulated by internal acidification. Both types of regulation are indirect. TWIK‐1 channel activity is blocked by Ba2+(IC50=100 microM), quinine (IC50=50 microM) and quinidine (IC50=95 microM). This channel is of particular interest because its mRNA is widely distributed in human tissues, and is particularly abundant in brain and heart. TWIK‐1 channels are probably involved in the control of background K+ membrane conductances.

We report the cloning of a mouse GIRK2 splice variant, noted mGIRK2A. Both channel proteins are functionally expressed in Xenopus oocytes upon injection of their cRNA, alone or in combination with the GIRK1 cRNA. Three GIRK channels, mGIRK1-3, are shown to be present in the brain. Colocalization in the same neurons of mGIRK1 and mGIRK2 supports the hypothesis that native channels are made by an heteromeric subunit assembly. GIRK3 channels have not been expressed successfully, even in the presence of the other types of subunits. However, GIRK3 chimeras with the aminoand carboxyl-terminal of GIRK2 are functionally expressed in the presence of GIRK1. The expressed mGIRK2 and mGIRK1, -2 currents are blocked by Ba 2؉and Cs ؉ ions. They are not regulated by protein kinase A and protein kinase C. Channel activity runs down in inside-out excised patches, and ATP is required to prevent this rundown. Since the nonhydrolyzable ATP analog AMP-PCP is also active and since addition of kinases A and C as well as alkaline phosphatase does not modify the ATP effect, it is concluded that ATP hydrolysis is not required. An ATP binding process appears to be essential for maintaining a functional state of the neuronal inward rectifier K ؉ channel. A Na ؉ binding site on the cytoplasmic face of the membrane acts in synergy with the ATP binding site to stabilize channel activity.

MbIRK3, mbGIRK2 and mbGIRK3 K' channels cDNAs have been cloned from adult mourn brain. These cDNAs encode polypeptides of 445,414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane-spanning domains Ml and M2 and a pore-forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMKl and GIRKl. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRKl (60%) than to ROMKl and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K+-selective currents very similar to IRK1 and GIRKl , respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G-protein coupled inward rectityer K' channels which has been observed through their functional coupling with 1.

This work describes the cloning and expression of the levocabastine-sensitive neurotensin (NT) receptor from mouse brain. The receptor protein comprises 417 amino acids and bears the characteristics of G-protein-coupled receptors. This new NT receptor (NTR) type is 39% homologous to, but pharmacologically distinct from, the only other NTR cloned to date from the rat brain and the human HT29 cell line. When the receptor is expressed in Xenopus laevis oocytes, the H1 antihistaminic drug levocabastine, like NT and neuromedin N, triggers an inward current. The pharmacological properties of this receptor correspond to those of the low-affinity, levocabastine-sensitive NT binding site described initially in membranes prepared from rat and mouse brain. It is expressed maximally in the cerebellum, hippocampus, piriform cortex, and neocortex of adult mouse brain.

Potassium channels have an essential role in repolarization phases of action potentials and in the fine regulation of the resting potential. Molecular cloning has recently led to the identification of a large number (over 15) of genes for voltagesensitive, non inward-rectifier, K ϩ (Kv) channels (1, 2) which, when expressed in Xenopus oocytes, generate a variety of K ϩ channel activities with different kinetics, voltage dependences, conductances, and regulation properties. Surprisingly, only a relatively small number of toxins active on these channels has yet been discovered (3, 4). They are MCD peptide from bee venom (5, 6), charybdotoxin and analogs from different scorpion species (7-14), ␤-bungarotoxin (15, 16), and dendrotoxins from mamba venoms (3,5,(17)(18)(19)(20)(21)(22).These different toxins only block the expression of four of the cloned Kv channels (Kv1.1, Kv1.2, and Kv1.6 for MCD peptide and dendrotoxin, Kv1.1, Kv1.2, Kv1.3, and Kv1.6 for charybdotoxin) (reviewed in Ref. 23). Binding studies using radioiodinated derivatives of these toxins have been essential for the identification, purification, and determination of the subunit structure (6, 24 -26) of these Kv channels. These toxins have also been important for the first brain localizations of Kv channels (16,27) and are particularly interesting inducers of long term potentiation (28).Sea anemones produce toxins with which they paralyze their prey. They are particularly important as sources of toxins active on voltage-dependent Na ϩ channel which have been essential tools for studying the structure, the mechanism, and the diversity of this channel type (29 -38).This paper reports the isolation, structure, and properties of a series of new toxins from Anemonia sulcata which behave as blockers of Kv channels. EXPERIMENTAL PROCEDURES Materials-Trypsin, the Kunitz trypsin inhibitor (BPTI),1 and N ␣ -benzoyl-DL-arginine p-nitroanilide (BAPNA) were obtained from Sigma. Sephadex G-25, Sephadex G-50, SP Sephadex C-25 were obtained from Pharmacia, Fractogel TSK HW-50 (F), Fractogel EMD SO 3 -650 (M), and RP18 Lichrocart were from Merck. For HPLC columns, TSK SP 5PW was from Toyosoda. Ultrasphere ODS was from Beckman, Hypersil BDS was from SFCC Shandon, and Alltima was from Alltech. HPLC purifications were performed with a Waters system.Purification of Anemonia Sulcata Peptides-The first steps of this purification were performed with slight modifications of a method previously described for the isolation of Na ϩ channel toxins of A. sulcata (39). In this procedure 12 g of the crude sea anemone toxin (Ref. 39; Fig. 2B1) was dissolved in 120 ml of NaCl 1 M and regelfiltered in two parts on a Sephadex G-50 medium column (7 ϫ 140 cm) equilibrated in 1 M NaCl. The crab paralyzing fractions of these gel filtrations were combined, dialyzed in a Visking Dialysis tube (molecular weight cutoff 12,000 -14,000) for 5 h, concentrated at reduced pressure, and desalted on a Sephadex G-25 column (7 ϫ 70 cm) equilibrated with 0.3 M acetic acid. After a concentration at red...

Outward rectifier K+ channels have a characteristic structure with six transmembrane segments and one pore region. A new member of this family of transmembrane proteins has been cloned and called Kv8.1. Kv8.1 is essentially present in the brain where it is located mainly in layers II, IV and VI of the cerebral cortex, in hippocampus, in CA1‐CA4 pyramidal cell layer as well in granule cells of the dentate gyrus, in the granule cell layer and in the Purkinje cell layer of the cerebellum. The Kv8.1 gene is in the 8q22.3–8q24.1 region of the human genome. Although Kv8.1 has the hallmarks of functional subunits of outward rectifier K+ channels, injection of its cRNA in Xenopus oocytes does not produce K+ currents. However Kv8.1 abolishes the functional expression of members of the Kv2 and Kv3 subfamilies, suggesting that the functional role of Kv8.1 might be to inhibit the function of a particular class of outward rectifier K+ channel types. Immunoprecipitation studies have demonstrated that inhibition occurs by formation of heteropolymeric channels, and results obtained with Kv8.1 chimeras have indicated that association of Kv8.1 with other types of subunits is via its N‐terminal domain.

TWIK‐1 is a new type of K+ channel with two P domains and is abundantly expressed in human heart and brain. Here we show that TWIK‐1 subunits can self‐associate to give dimers containing an interchain disulfide bridge. This assembly involves a 34 amino acid domain that is localized to the extracellular M1P1 linker loop. Cysteine 69 which is part of this interacting domain is implicated in the formation of the disulfide bond. Replacing this cysteine with a serine residue results in the loss of functional K+ channel expression. This is the first example of a covalent association of functional subunits in voltage‐sensitive channels via a disulfide bridge.