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Screening and cDNA Cloning of Kv1 Potassium Channel Toxins in Sea Anemones

When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of 125I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3′RACE and 5′RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ1.3-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34–63% identities with the other type 1 potassium channel toxins.

It should be noted that all the sea anemones that have been shown to contain potassium channel toxins, including Kv1 channel toxins, belong to either the family Actiniidae or the family Stichodactylidae. Our previous screening failed to detect Kv1 potassium channel toxins in any of the five species of the families differing from the above two families [22]. In this study, therefore, screening for Kv1 potassium channel toxins, which was based on the competitive inhibition of the binding of radiolabeled α-dendrotoxin (Kv1 potassium channel toxin from the green mamba Dendroaspis angusticeps [23]) to rat synaptosomal membranes, was further extended to 21 species of sea anemones in seven families. Furthermore, molecular cloning was attempted to elucidate the primary structures of type 1 potassium channel toxins, for which degenerate primers could be designed from the known nucleotide sequences of the cDNAs encoding κ 1.3 -ATTX-Aer1a [12] and κ 1.3 -SHTX-Hm1a [15].

Screening of potassium channel toxins
Crude extracts from 21 species of sea anemones were examined for Kv1 potassium channel toxicity by competitive inhibition experiments. As shown in Figure 1, inhibition of the binding of 125 I-α-dendrotoxin to rat synaptosomal membranes was observed in all species with varied potencies.
The species with only weak inhibitory activity were considered to be hardly selected as samples in future study on potassium channel toxins. In this study, therefore, the following 11 species showing more than 50% inhibition were judged to be substantially positive: two species (Macrodactyla doreensis and Telactinia citrina) of the family Actiniidae, one species (Calliactis polypus) of the family Hormathiidae, five species (Heteractis magnifica, Mesactinia ganensis, Stichodactyla haddoni, Stichodactyla mertensii and Stichodactyla tapetum) of the family Stichodactylidae and three species (Cryptodendrum adhaesivum, Heterodactyla hemprichii and Thalassianthus aster) of the family Thalassianthidae. One Kv1 potassium channel toxin (κ 1.3 -SHTX-Hm1a) has already been isolated from H. magnifica [15] and three Kv1 potassium channel toxins (κ 1.3 -SHTX-Sha2a, κ 1.3 -SHTX-Sha3a and κ 1.3 -SHTX-Sha3b) from S. haddoni [17]. Furthermore, previous screening has established the occurrence of Kv1 potassium channel toxins in S. mertensii [21]. The remaining eight species were first demonstrated to be positive in this study. So far, Kv1 potassium channel toxins have not been found in any species other than those belonging to the family Actiniidae or Stichodactylidae. In view of this, our screening data are of particular value in showing the occurrence of Kv1 potassium channel toxins in one species of Hormathiidae and three species of Thalassianthidae. Based on our results and previous data, distribution of Kv1 potassium channel toxins in sea anemones is summarized in Table 1. Of the 44 species examined, 18 species belonging to four families (Actiniidae, Hormathiidae, Stichodactylidae and Thalassianthidae) contain Kv1 potassium channel toxins. In general, sodium channel toxins are lethal to crustaceans. On the other hand, potassium channel toxins are not lethal to crustaceans, although some of them, such as three toxins (κ 1.3 -SHTX-Sha2a, κ 1.3 -SHTX-Sha3a and κ 1.3 -SHTX-Sha3b) from S. haddoni [17], are paralytic.
To our experience, crude extracts from various sea anemones are lethal to freshwater crabs (Potamon dehaani) without exception, indicating a ubiquitous distribution of sodium channel toxins in sea anemones. Therefore, the distribution of Kv1 potassium channel toxins in sea anemones is considerably wide but seems to be narrower than that of sodium channel toxins.
Five species of the genus Stichodactyla in the family Stichodactylidae are all positive, suggesting the common occurrence of Kv1 potassium channel toxins in this genus. Similarly, three species of the family Thalassianthidae are all positive, although they are classified into different genera. It is likely that members of Thalassianthidae commonly contain Kv1 potassium channel toxins. In addition, Kv1 potassium channel toxins might be widely distributed in members of the three genera (Actinia, Anemonia and Bunodosoma) in the family Actiniidae, although only two species in each genus have been tested and found to be positive. Further screening experiments using much more species are needed to confirm the relationships between the occurrence of Kv1 potassium channel toxins and the taxonomical position of sea anemones. [21] [14,21] This study [22] [22] This study [22] This study Actinodendronidae  This study [22] This study This study [22] This study [15], This study This study This study a [17], This study [16,21] [

Cloning of cDNAs encoding type 1 potassium channel toxins
RT-PCR using a pair of degenerate primers (RT-f and RT-r; refer to Table 2 for the nucleotide sequences of the primers) was performed for the following eight species: the six positive species (M. doreensis, S. haddoni, S. mertensii, C. adhaesivum, H. hemprichii and T. aster) found in this study, A. equina previously shown to have a potassium channel toxin (κ 1.3 -ATTX-Aeq1a) [11] and S. gigantea (a member of the genus Stichodactyla) strongly assumed to have a potassium channel toxin. Amplified products were obtained for three Stichodactyla species (S. gigantea, S. haddoni and S. mertensii) of the family Stichodactylidae and three species (C. adhaesivum, H. hemprichii and T. aster) of the family Thalassianthidae but not for two species (A. equina and M. doreensis) of the family Actiniidae. As expected, approximately 150 bp products were amplified for S. haddoni, C. adhaesivum and H. hemprichii. However, longer products (approximately 350 bp), corresponding to the region between the forward primer (RT-f) position and the 3′-end, were amplified for S. gigantea, S. mertensii and T. aster, probably because the degenerate reverse primer (RT-r) did not anneal to the template. Therefore, 3′RACE to analyze the nucleotide sequences of the 3′-terminal regions was carried out only for S. haddoni, C. adhaesivum and H. hemprichii. Finally, nucleotide sequences of the 5′-terminal regions were determined by 5′RACE.
After subcloning each PCR product into the pT7Blue T-vector, at least three clones were analyzed for nucleotide sequence. For each PCR product, there was no difference in nucleotide sequence among the clones analyzed, suggesting that isoforms of the cloned toxin, if present, are trace in the six species. The determined nucleotide sequences of the full-length cDNAs coding for six type 1 potassium channel toxins (named κ 1.
Type 1 potassium channel toxins can be further divided into subtype 1a and 1b toxins, having four and eight amino acid residues, respectively, between the second and third Cys residues from the N-terminus [12], although little is understood as to how the structural difference between these subtypes is related to the potassium channel toxicity. The six toxins cloned in this study are apparently subtype 1a toxins. It is interesting to note that the distribution of subtype 1a and 1b toxins is associated with the taxonomical position of sea anemones. Eight subtype 1a toxins (κ 1.3 -SHTX-Sg1a, κ 1.3 -SHTX-Sha1a, κ 1.3 -SHTX-Sm1a, κ 1.3 -SHTX-She1a, κ 1.3 -SHTX-Hm1a, κ 1.3 -TLTX-Ca1a, κ 1.3 -TLTX-Hh1a and κ 1.3 -TLTX-Ta1a) are contained in members of the family Stichodactylidae or Thalassianthidae and three subtype 1b toxins (κ 1.3 -ATTX-Aeq1a, κ 1.3 -ATTX-As1a and κ 1.3 -ATTX-Bg1a) in those of the family Actiniidae; the only exception is κ 1.3 -ATTX-Aer1a, a subtype 1a toxin in A. erythraea (member of the family Actiniidae). At present, no information about the amino acid sequences of subtype 1b toxin precursors is available. In this study, neither κ 1.3 -ATTX-Aeq1a (subtype 1b toxin of A. equina) nor a type 1 toxin (presumably subtype 1b toxin) of M. doreensis (member of Actiniidae) could be cloned, since no amplified products were obtained by RT-PCR using the degenerate primers (RT-f and RT-r). Therefore, molecular cloning of κ 1.3 -ATTX-Aeq1a using degenerate primers designed from its amino acid sequence is now under study.
It is apparent that the six type 1 potassium channel toxins (κ 1.3 -SHTX-Sg1a, κ 1.3 -SHTX-Sha1a, κ 1.3 -SHTX-Sm1a, κ 1.3 -TLTX-Ca1a, κ 1.3 -TLTX-Hh1a and κ 1.3 -TLTX-Ta1a) are individually expressed in the six species of sea anemones, since their cDNAs were cloned using total RNA as the starting material. However, the potassium channel toxicity detected in each sea anemone by competitive inhibition experiments using 125 I-α-dendrotoxin may not be explained only by the type 1 toxin cloned. Indeed, one type 2 toxin (κ 1.3 -SHTX-Sha2a) and two type 4 toxins (κ 1.3 -SHTX-Sha3a and b) had been found in S. haddoni [17] from which κ 1.3 -SHTX-Sha1a was cloned in this study. Future study is needed to isolate and characterize potassium channel toxins from the positive species. Such study might discover structurally and functionally novel potassium channel toxins. Asterisks under the sequence of κ 1.3 -ATTX-Bg1a represent the dyad (Lys-Tyr) that is crucial for the binding to potassium channels.

Sea anemone samples
The following 23 species of sea anemones belonging to seven families were used in this study:  Table 2 for their taxonomical position). Except for two species (A. equina and S. gigantea used only for cDNA cloning), 21 species were used for screening of Kv1 potassium channel toxins. Specimens of A. equina were collected at Katsuura, Chiba Prefecture; those of B. mcmurrichi and A. maculata at Shishijima, Kagoshima Prefecture; those of T. clavata at Okinoshima, Chiba Prefecture; and those of C. polypus along the coast of Ishigaki Island, Okinawa Prefecture. These specimens were transported frozen or alive to our laboratory. For the remaining species, live specimens were purchased from retail aquarium shops. Most of the specimens were stored at −20 °C until extraction. For cDNA cloning, one live specimen of each species was cut into small pieces, which were immediately frozen in liquid nitrogen and stored at −80 °C until use.

Preparation of crude extracts
Each frozen sample was well macerated and 2-3 g of the macerate was homogenized in five volumes of distilled water. After centrifugation, the supernatant obtained was used as a crude extract.

Assay of potassium channel toxicity
Potassium channel toxicity was indirectly assayed by competitive inhibition of the binding of 125 I-α-dendrotoxin to rat synaptosomal membranes, as reported previously [11,33]. Synaptosomal membrane suspension (0.4 mg protein/mL) was prepared from rat brains (Funakoshi, Tokyo, Japan).

Conclusions
Kv1 potassium channel toxins are widely distributed in sea anemones, including not only members of the two families (Actiniidae and Stichodactylidae) but also of other families such as Thalassianthidae. Based on the amino acid sequence features, type 1 potassium channel toxins are divided into two subtypes (subtypes 1a and 1b). Subtype 1a toxins are contained in members of Stichodactylidae and Thalassianthidae and subtype 1b toxins in Actiniidae; the only exception is κ 1.3 -ATTX-Aer1a, a subtype 1a toxin in Anemonia erythraea (member of Actiniidae).