Predictive impact of rare genomic copy number variations in siblings of individuals with autism spectrum disorders.

Identification of genetic biomarkers associated with autism spectrum disorders (ASDs) could improve recurrence prediction for families with a child with ASD. Here, we describe clinical microarray findings for 253 longitudinally phenotyped ASD families from the Baby Siblings Research Consortium (BSRC), encompassing 288 infant siblings. By age 3, 103 siblings (35.8%) were diagnosed with ASD and 54 (18.8%) were developing atypically. Thirteen siblings have copy number variants (CNVs) involving ASD-relevant genes: 6 with ASD, 5 atypically developing, and 2 typically developing. Within these families, an ASD-related CNV in a sibling has a positive predictive value (PPV) for ASD or atypical development of 0.83; the Simons Simplex Collection of ASD families shows similar PPVs. Polygenic risk analyses suggest that common genetic variants may also contribute to ASD. CNV findings would have been pre-symptomatically predictive of ASD or atypical development in 11 (7%) of the 157 BSRC siblings who were eventually diagnosed clinically.

CNTN6 mutations are risk factors for abnormal auditory sensory perception in autism spectrum disorders.

Contactin genes CNTN5 and CNTN6 code for neuronal cell adhesion molecules that promote neurite outgrowth in sensory-motor neuronal pathways. Mutations of CNTN5 and CNTN6 have previously been reported in individuals with autism spectrum disorders (ASDs), but very little is known on their prevalence and clinical impact. In this study, we identified CNTN5 and CNTN6 deleterious variants in individuals with ASD. Among the carriers, a girl with ASD and attention-deficit/hyperactivity disorder was carrying five copies of CNTN5. For CNTN6, both deletions (6/1534 ASD vs 1/8936 controls; P=0.00006) and private coding sequence variants (18/501 ASD vs 535/33480 controls; P=0.0005) were enriched in individuals with ASD. Among the rare CNTN6 variants, two deletions were transmitted by fathers diagnosed with ASD, one stop mutation CNTN6W923X was transmitted by a mother to her two sons with ASD and one variant CNTN6P770L was found de novo in a boy with ASD. Clinical investigations of the patients carrying CNTN5 or CNTN6 variants showed that they were hypersensitive to sounds (a condition called hyperacusis) and displayed changes in wave latency within the auditory pathway. These results reinforce the hypothesis of abnormal neuronal connectivity in the pathophysiology of ASD and shed new light on the genes that increase risk for abnormal sensory perception in ASD.

Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation.

BACKGROUND: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a 'patchiness' will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development. METHODS: XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements. RESULTS: Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR). CONCLUSIONS: The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed.

PP065. dNK and dNK-CM mediated alterations of DNA methylation in extravillous cytotrophoblasts (EVTS).

INTRODUCTION: Placental DNA methylation is thought to be influenced by environmental exposure. Decidual natural killer cells (dNKs) directly contact with cytotrophoblasts in the early stage of pregnancy. dNKs may affect DNA methylation of extravillous cytotrophoblasts (EVTs) directly or indirectly through their secreted soluble factors. OBJECTIVES: Previously, we showed that EVT outgrowth and migration on collagen gel were restricted by exposure to dNK or dNK-derived conditioned medium (dNK-CM) (ref [1]). The aim of this study was to determine if EVT DNA methylation was altered by treating with dNK or dNK-CM. METHODS: Placental explants collected from 6 first-trimester healthy pregnancy terminations were cultured on a rat collagen gel model. Outgrowth EVTs from each subject were treated with medium or concordant dNK (trapped inside of hollow fibers) or dNK-CM. EVTs were harvested after 96-hour co-culturing and underwent DNA extraction. DNA methylation was quantified using the Infinium Human Methylation 450 BeadChip, which targets over 450,000 CpG sites in the human genome. Differential methylation was defined by having p<0.05 (Student's t-test) and average DNA methylation change >10% before and after treatments. Functional enrichment was assessed by gene ontology analysis with False Discovery Rate <10% defined as significantly enriched. RESULTS: Increased DNA methylation was observed for 360 loci and 572 loci by dNK or dNK-CM respectively, and decreased DNA methylation was shown for 62 loci and 188 loci by dNK or dNK-CM respectively. DNA methylation at 44 loci was altered by both dNK and dNK-CM. The common loci were overrepresented for associations with EVT differentiation, adhesion and migration. Examples of the relevant overlapped loci with increased DNA methylations were MYO15A and PRDM16 (PR domain zinc finger protein 16); and the overlapped loci with reduced DNA methylation were CDH9 and USP29 (ubiquitin specific protein 29). dNK but not dNK-CM reduced IL18 methylation and increased methylation on ITGAL (integrin, alpha L) and ITGB7. dNK-CM but not dNK reduced methylation of ITGAD and PCDH8 (protocadherin 8) and increased methylation of CDH4 and CDH6. CONCLUSION: DNA methylation of EVT was altered by exposure to surrounding dNK and their secreted soluble molecules. These results serve as a basis for further investigations on whether DNA methylation can mediate the changes in protein expression that influence EVT differentiation, adhesion and migration.

Review: A high capacity of the human placenta for genetic and epigenetic variation: implications for assessing pregnancy outcome.

Genetic and epigenetic studies of the human placenta can help to clarify the underlying mechanisms of placenta-associated diseases. However, such studies have also revealed a considerable degree of within- and between-placenta variability, which can be attributed to a variety of influences. We illustrate the inherent heterogeneity in the placenta using examples from two types of studies: 1) chromosomal mosaicism and 2) DNA methylation variation. We discuss the factors that may influence the distribution of variation and how, understanding the source of this variation is important for interpreting data used to investigate and predict clinical outcomes.

IFPA Meeting 2010 Workshop Report I: Immunology; ion transport; epigenetics; vascular reactivity; epitheliochorial placentation; proteomics.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

The utility of quantitative methylation assays at imprinted genes for the diagnosis of fetal and placental disorders.

An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology.

Evaluating DNA methylation and gene expression variability in the human term placenta.

UNLABELLED: Obtaining representative samples from a term placenta for gene-expression studies is confounded by both within placental heterogeneity and sampling effects such as sample location and processing time. Epigenetic processes involved in the regulation of gene expression, such as DNA methylation, may show similar variability, but are less well studied. Therefore, we investigated the nature of within and between- placenta variation in gene expression and DNA methylation of genes that were chosen for being differentially expressed or methylated by cell type within the placenta. METHODS: In total, two or more samples from each of 38 normal term placentae were utilized. The expression levels of CDH1, CDH11, ID2, PLAC1 and KISS1 were evaluated by real-time PCR. DNA methylation levels of LINE1 elements and CpGs within the promoter regions of KISS1, PTPN6, CASP8, and APC were similarly quantified by pyrosequencing. RESULTS: Despite considerable sample-to-sample variability within each placenta, the within-placenta correlation for both gene expression and methylation was significant for each studied gene. Most of this variability was not due to sample location. However, between placental differences in gene expression were inflated by the dramatic effect of processing time (0-24 h) on mRNA levels, particularly for PLAC1 and KISS1 (both expressed in the apical syncytiotrophoblast). In contrast, DNA methylation levels remained relatively constant over this same time period. CONCLUSION: Due to extensive site-to-site variability, multiple sampled sites are needed to accurately represent a placenta for molecular studies. Furthermore, mRNA quantitation of some genes may be hampered by its rapid degradation post-delivery (and possibly perinatally) and thus processing time should be considered in such analyses. Within-placenta correlations in expression and methylation from unrelated genes raise the possibility that methylation and expression variation may potentially reflect cell composition differences between samples rather than true differences occurring at the cellular level.

Proton driven vase-to-kite conformational change in cavitands at an air-water interface monitored by surface SHG.

The conformational changes of quinoxaline-bridged cavitands deposited as Langmuir films were monitored at different pH values of the subphase using surface second harmonic generation during the compression of the monolayer at the water surface. A quantitative analysis of the susceptibility tensor elements was performed for methylene (MeCav)- and quinoxaline (QxCav)-bridged cavitands for pH values varying between 5.7 and 0.1. For MeCav (reference compound), no significant changes were observed for different pHs, confirming that the cavity does not undergo protonation or a drastic conformational change. For the QxCav, however, the results suggest a partial opening of the cavity on the basis of analysis of the compression curves.

Role of free thiol groups in the biological activities of stonustoxin, a lethal factor from stonefish (Synanceja horrida) venom.

Stonustoxin (SNTX) is a two-subunit protein purified from the venom of a stonefish, Synanceia horrida. It has potent lethal activity and is also a membrane pore-forming cytolysin. The role of thiol groups in the biological activities of SNTX was investigated. Both the hemolytic and lethal activities of SNTX were potentiated by the reducing agent, dithiothreitol (DTT). The hemolytic activity of SNTX was sensitive to the modification of thiol groups by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The loss of haemolytic activity correlated with the number of thiol groups that were titrated with DTNB. Thiol modification of SNTX with DTNB also inhibited its lethality. These inhibitory effects of thiol modification could be reversed by reduction with DTT. It was also found that the haemolytic activity of SNTX could not be inhibited by cholesterol. These observations indicate that free thiol groups play an important role in the haemolytic activity and lethality of SNTX but unlike other thiol-activated cytolysins, SNTX was not inhibited by cholesterol. Thus, SNTX may represent a new class of cytolytic toxin.

The role of cationic amino acid residues in the lethal activity of stonustoxin from stonefish (Synanceja horrida) venom.

Stonustoxin (SNTX) is a two subunit pore-forming cytolytic protein purified from the venom of the stonefish (Synanceja horrida). SNTX also possesses lethal activity. Since cationic residues contribute significantly to the cytolytic activity of several pore-forming toxins, we examined the role of lysine and arginine residues in the lethal activity of SNTX. SNTX lost its lethal activity when the positively-charged side chains of lysine residues were converted to negatively-charged side chains upon succinylation. When the arginine residues were modified using 2,3-butanedione, SNTX also lost its lethal activity. However, the domains for cytolytic and lethal activity may not necessarily be the same.

Haemolytic activity of stonustoxin from stonefish (Synanceja horrida) venom: pore formation and the role of cationic amino acid residues.

Stonustoxin (SNTX) is a two-subunit protein toxin purified from the venom of the stonefish (Synanceja horrida), which induces potent haemolytic activity. We examined the pore-forming property of this non-enzymic protein by an osmotic protection assay. SNTX-induced haemolysis was completely prevented by osmotic protectants of adequate size [poly(ethylene) glycol 3000; molecular diameter approx. 3.2 nm]. Uncharged molecules of smaller size, such as raffinose and poly(ethylene) glycol 1000-2000, failed to protect against cell lysis. These findings indicate that SNTX induces the formation of hydrophilic pores in the cell membrane, which results in the lysis of erythrocytes. Since cationic residues contribute significantly to the cytolytic activity of several other pore-forming toxins, we examined the role of positively charged lysine and arginine residues in the haemolytic activity of SNTX. SNTX lost its haemolytic activity when the positively charged side chains of lysine residues were neutralized or converted into negatively charged side chains upon carbamylation or succinylation respectively. The haemolytic activity of SNTX was also inhibited by the modification of positively charged arginine residues using 2,3-butanedione. The loss of haemolysis showed strong correlation with the number of Lys or Arg residues modified. CD analyses, however, showed that the conformation of SNTX was not significantly affected by these chemical modifications. Further, the haemolytic activity of SNTX was competitively inhibited by various negatively charged lipids, such as phosphatidylserine, cardiolipin and monosialogangliosides. These results indicate that SNTX induces potent haemolytic activity through the formation of pores in the cell membrane, and that cationic residues play a crucial role in its cytolytic mechanism.

Effects of thymosin alpha1 on the development of amoeboid microglial cells in the corpus callosum of neonatal BALB/c and athymic mice.

The present study investigated the effects of intraperitoneal injections of thymosin alpha1 on the supraventricular amoeboid microglial cells (SAMC) in the newborn athymic and normal BALB/c mice. The microglial cells labelled by the lectin GSA I-B4 and the antibody Mac-1 showed a 27% reduction in number in the athymic mice receiving thymosin alpha1 injections compared with those receiving vehicle injections, and a 37% reduction in BALB/c mice receiving thymosin alpha1 injections compared with those receiving vehicle injections. Some of the SAMC in both BALB/c and athymic mice receiving thymosin alpha1 injections became ramified, while the remainder still exhibited their normal amoeboid appearance with few filopodial processes. Ultrastructurally, the lectin reaction product was confined to the plasma membrane and some cytoplasmic vacuoles of labelled SAMC. In both BALB/c and athymic mice, some labelled microglial cells became slender or elongated after thymosin alpha1 injections. Also their cytoplasm was reduced and contained fewer organelles. Radioimmunoassay of the plasma of thymosin alpha1 and vehicle-injected mice showed that there was a significant increase in the cortisol level in BALB/c (P < 0.01) and athymic (P < 0.001) mice 5 days after thymosin alpha1 injections, compared with that of the control mice. The results point to a strong correlation between the reduction of SAMC and the increased level of plasma cortisol. Supporting this is the fact that cortisol is known to suppress the production of monocytes considered to be the precursors of amoeboid microglia.

Stimulation of haemolytic activity of sea anemone cytolysins by 8-anilino-1-naphthalenesulphonate.

This study reports for the first time stimulation of protein activity by the hydrophobic probe, 8-anilino-1-naphthalenesulphonate (ANS). Magnificalysin (HMg) I and II and equinatoxin (EqTx) II and III are cytolysins isolated from the sea anemone Heteractis magnifica and Actinia equina, respectively. The haemolytic activity of these cytolysins could be stimulated by treatment with ANS. Their activation involved conformational changes following ANS treatment as shown by fluorescence spectra. ANS-induced conformational changes were reversible upon removal of ANS. ANS-stimulated activity of HMg I was inhibited by sphingomyelin and antiserum but not affected by bromosuccinimide (NBS) which oxidises tryptophan residues. However, toxin pre-treated with NBS could no longer be stimulated by addition of ANS. Energy transfer from tryptophan to ANS was observed by a fluorescence scan. Hence the tryptophan residues appear to be involved, at least partially, in ANS-binding. ANS-induced conformational change may be responsible for the activation of the cytolytic activity of these cytolysins.

Stonustoxin is a novel lethal factor from stonefish (Synanceja horrida) venom. cDNA cloning and characterization.

Stonustoxin (SNTX) is a multifunctional lethal protein isolated from venom elaborated by the stonefish, Synanceja horrida. It comprises two subunits, termed alpha and beta, which have respective molecular masses of 71 and 79 kDa. SNTX elicits an array of biological responses both in vitro and in vivo, particularly a potent hypotension that appears to be mediated by the nitric oxide pathway. As a prelude to structure-function studies, we have isolated and sequenced cDNA clones encoding the alpha- and beta-subunits of SNTX from a venom gland cDNA library. The deduced amino acid sequence of neither subunit shows significant homology with any known protein. Protein sequence alignment does, however, show the subunits to be 50% homologous to each other and implies that they may have arisen from a common ancestor. The subunits of this novel toxin lack typical N-terminal signal sequences commonly found in proteins that are secreted via the endoplasmic reticulum-Golgi apparatus pathway, indicating the possibility of its being secreted by a non-classical pathway, which is not clearly understood. The SNTX subunits have been expressed in Escherichia coli as cleavable fusion proteins that cross-react with antibodies raised against the native toxin. To the best of our knowledge, this is the first complete sequence of a fish-derived protein toxin to be reported.

Primary angiitis of the central nervous system associated with Hodgkin's disease.

An unusual case of primary angiitis of the central nervous system associated with Hodgkin's disease in a 55-year-old-man is reported. After a 10-month history of acute transverse myelitis, the patient was diagnosed as having nodular sclerosing-type Hodgkin's disease involving the retroperitoneal lymph nodes. The patient died of a brainstem hemorrhage 1 week after a 15-day course of chemotherapy. Primary angiitis was documented on autopsy examination. To our knowledge, only nine similar cases have been reported in the literature, and none of them was associated with a sole initial spinal cord presentation. Owing to the rarity of this disease entity, a high index of suspicion and awareness of the association between primary angiitis and Hodgkin's disease are essential for early diagnosis.

Inhibition of sodium-dependent uptake processes in purified rat brain synaptosomes by Lophozozymus pictor toxin and palytoxin.

To get an insight into the mechanism of neurotoxicity exhibited by Lophozozymus pictor toxin (LPTX) and the toxin isolated from P.caribaeorum (C-PTX) studies were carried out on the effect of these toxins on the uptake of selected substrates (neurotransmitters, amino acids and glucose) in isolated nerve endings. The toxins were found to inhibit the uptake of gamma-aminobutyric acid (GABA), noradrenaline, choline, L-leucine and 2-deoxy-D-glucose in rat brain synaptosomes. LPTX- or C-PTX-induced inhibition of synaptosomal uptake was reduced in the absence of Na+ in the assay medium. Synaptosomes exposed to LPTX and C-PTX release K+ in a dose-dependent manner. Ouabain, a selective inhibitor of the plasma membrane Na+, K(+)-ATPase could inhibit LPTX- and C-PTX-induced K+ efflux from synaptosomes and alleviate the toxin-induced inhibition of synaptosomal GABA uptake. It appears that the induction of ionic flux is the primary cause of toxicity by these toxins leading to the inhibition of Na(+)-dependent uptake processes in synaptosomes. The antagonistic action of ouabain suggests the involvement of the membrane sodium pump in the development of cytotoxicity.

The chemical composition of gallstones: its relevance to surgeons in Southeast Asia.

This paper describes the methods in the study of the chemical composition of gallstones and the application of this knowledge to the practising surgeon in Southeast Asia. There are three types of stone in this part of the world. Inspecting with the naked eye, the surgeon is usually able to recognize accurately the stone type--cholesterol, black pigment and brown pigment. The stone type together with the clinicopathological and radiological findings enable the surgeon to make a confident diagnosis of either the Western-type (cholesterol and black pigment) or Asiatic-type (brown pigment) gallstone disease in each patient, and based on this, a rational approach in the management of the patient can be instituted. In a personal series of 484 cases, cholesterol stones formed 46% of the cases, black pigment stones 30.5% and brown stones 13%; and cholesterosis and acalculous cholecystitis constituted the remaining 10.5%. In rapidly developing Singapore, Western-type cholesterol stones now predominate while brown stones appear to be decreasing. In patients with small bile duct stones of any stone type, endoscopic extraction followed by laparoscopic cholecystectomy appears to be the procedure of choice. Large bile duct stones require open surgery. In patients with grossly dilated bile ducts containing brown stones, biliary enteric bypass is performed to reduce biliary stasis, cholangitis and recurrent stone formation.

Synaptosomal binding of 125I-labelled daboiatoxin, a new PLA2 neurotoxin from the venom of Daboia russelli siamensis.

Daboiatoxin (DbTx), the PLA2 neurotoxin from Daboia russelli siamensis venom, was shown to bind specifically and saturably to rat cerebrocortical synaptosomes and synaptic membrane fragments. Two families of binding sites were detected by equilibrium binding analysis in the presence and absence of Ca2+. Scatchard analysis of biphasic plateaus revealed Kdl 5 nM and Bmax1, 6 pmoles/mg protein, and Kd2 80 nM and Bmax2 20 pmoles/mg protein, respectively, for the high- and low-affinity binding sites. The binding of 125I-DbTx to synaptosomes did not show marked dependence on Ca2+, Mg2+, Co2+ and Sr2+. Native DbTx was the only strong competitor to 125I-DbTx synaptosomal binding (IC50 12.5 nM, KI 5.5 nM). Two other crotalid PLA2 neurotoxins, crotoxin CB and mojave toxin basic subunit, and nontoxic C. Atrox PLA2 enzyme, were relatively weaker inhibitors, while two viperid PLA2 neurotoxins, ammodytoxin A and VRV PL V, were very weak inhibitors. Crotoxin CA was a poor inhibitor even at microM concentrations, whereas no inhibitory effect at all was observed with crotoxin CACB, ammodytoxin C, VRV PL VIIIa, taipoxin, beta-bungarotoxin, or with PLA2 enzymes from N. naja venom, E. schistosa venom, bee venom and porcine pancreas. All other pharmacologically active ligands examined (epinephrine, norepinephrine, histamine, choline, dopamine, serotonin, GABA, naloxone, WB-4101, atropine, hexamethonium and alpha-bun-garotoxin) also failed to interfere with 125I-DbTx binding. As those competitors that showed partial inhibition were effective only at microM concentration range compared to the Kd (5 nM) of 125I-DbTx synaptosomal binding, DbTx could well recognize a different neuronal binding site. Rabbit anti-DbTx polyclonal antisera completely blocked the specific binding. When a range of Ca2+ and K+ channels modulators were examined, Ca2+ channel blockers (omega-conotoxins GVIA and MVIIC, taicatoxin, calciseptine and nitrendiprene) did not affect the binding even at high concentrations, while charybdotoxin was the only K+ channel effector that could partially displace 125I-DbTx synaptosomal binding amongst the K+ channel blockers tested (apamin, dendrotoxin-I, iberiotoxin, MCD-peptide, 4-aminopyridine and tetraethylammonium), suggesting that neither K+ nor Ca2+ channels are associated with DbTx binding sites.