Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H ؉ -gated Na ؉ channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligandgated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC 50 ‫؍‬ 0.9 nM) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.Proton-gated Na ϩ -permeable channels are the simplest form of ligand-gated channels. They are present in many neuronal cell types throughout the central nervous system (1-5), suggesting an important function of these channels in signal transduction associated with local pH variations during normal neuronal activity. These channels might also play an important role in pathological situations such as brain ischemia or epilepsy, which produce significant extracellular acidification. They are also present in nociceptive neurons (1-3, 5, 6) and are thought to be responsible for the sensation of pain that accompanies tissue acidosis in muscle and cardiac ischemia (7,8), corneal injury (9), and inflammation and local infection (10, 11). It is only very recently that the first proton-gated channel, acid-sensitive ion channel (ASIC) 1 was cloned (12). The ASICs belong to a superfamily that includes amiloride-sensitive epithelial Na ϩ channels (13, 14), the FMRFamide-gated Na ϩ channel (15), and the nematode degenerins (DEGs), which probably correspond to mechano-sensitive Na ϩ -permeable channels (16). Several ASIC subunits have now been described: ASIC1a (12), ASIC1b (17), ASIC2a (18 -21), ASIC2b (22), and ASIC3 (23-25). The different subunits produce channels with different kinetics, external pH sensitivities, and tissue distribution. They can form functional homomultimers as well as heteromultimers (21,22,26). ASIC1a and ASIC1b both mediate rapidly inactivating currents following rapid and modest acidification of the external pH. However, although ASIC1a is present in both brain and afferent sensory neurons, its splice variant ASIC1b is found only in sensory neurons (17). ASIC2a forms an active H ϩ -gated channel and is abundant in the brain but essentially absent in sensory neurons, whereas its splice variant ASIC2b is present in both brain an...

Acanthamoeba polyphaga mimivirus is the largest known virus in both particle size and genome complexity. Its 1.2-Mb genome encodes 911 proteins, among which only 298 have predicted functions. The composition of purified isolated virions was analyzed by using a combined electrophoresis/mass spectrometry approach allowing the identification of 114 proteins. Besides the expected major structural components, the viral particle packages 12 proteins unambiguously associated with transcriptional machinery, 3 proteins associated with DNA repair, and 2 topoisomerases. Other main functional categories represented in the virion include oxidative pathways and protein modification. More than half of the identified virion-associated proteins correspond to anonymous genes of unknown function, including 45 "ORFans." As demonstrated by both Western blotting and immunogold staining, some of these "ORFans," which lack any convincing similarity in the sequence databases, are endowed with antigenic properties. Thus, anonymous and unique genes constituting the majority of the mimivirus gene complement encode bona fide proteins that are likely to participate in well-integrated processes.Acanthamoeba polyphaga mimivirus (mimivirus) is the largest virus isolated so far (23). Based on its highly specific characteristics, this double-stranded-DNA icosahedral virus (47) is the first member of the new Mimiviridae family (33, 43). Computational annotation of its 1.2-Mb genome (33) revealed many atypical features, including the presence of key translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (of types IA, IB, and II) (2, 33). Another unique characteristic of mimivirus is the presence of nearly identical promoter sequence motifs upstream of half of its 911 proteinencoding genes (42), which are presumably associated with proteins expressed during the early or late-early phase. Only 23% of the predicted coding genes exhibit convincing homology to proteins of known function, and 39% of them do not exhibit a clear (E values, Ͻ10 Ϫ5 ) sequence database match (33). Such coding regions without sequence similarity to other genes in databases are considered orphan open reading frames (ORFs) and termed "ORFans" (12). The origin and function of ORFan genes are still a matter of controversy, with opinions ranging from considering them pieces of junk DNA (1,8,40,44) to seeing them as quickly evolving sequences encoding normally expressed functional proteins (38, 39). Recent clinical evidence raised the possibility that mimivirus might be a human pathogen causing pneumonia (4, 24, 34), as suspected when it was first isolated from a cooling tower following an outbreak of pneumonia (23).Mass spectrometry-based analysis has recently emerged as a technique of choice to identify more comprehensively the set of viral proteins associated with viral particles (19,29,49). We now present the application of this technique to the largest known, and presumably most complex, viral particle,...

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...

1 In the present study, two new peptides, phrixotoxins PaTx1 and PaTx2 (29 ± 31 amino acids), which potently block A-type potassium currents, have been puri®ed from the venom of the tarantula Phrixotrichus auratus. 2 Phrixotoxins speci®cally block Kv4.3 and Kv4.2 currents that underlie I to1 , with an 55IC 50 570 nM, by altering the gating properties of these channels. 3 Neither are the Shaker (Kv1), Shab (Kv2) and Shaw (Kv3) subfamilies of currents, nor HERG, KvLQT1/IsK, inhibited by phrixotoxins which appear speci®c of the Shal (Kv4) subfamily of currents and also block I to1 in isolated murine cardiomyocytes. 4 In order to evaluate the physiological consequences of the Ito1 inhibition, mice were injected intravenously with PaTx1, which resulted in numerous transient cardiac adverse reactions including the occurrence of premature ventricular beats, ventricular tachycardia and di erent degrees of atrioventricular block. 5 The analysis of the mouse electrocardiogram showed a dose-dependent prolongation of the QT interval, chosen as a surrogate marker for their ventricular repolarization, from 249+11 to 265+8 ms (P50.05). 6 It was concluded that phrixotoxins, are new and speci®c blockers of Kv4.3 and Kv4.2 potassium currents, and hence of I to1 that will enable further studies of Kv4.2 and Kv4.3 channel and/or I to1 expression.