Sylvie Diochot

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3‐containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino‐acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage‐sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 μM), ASIC1a+3 (IC50=2 μM), and no effect on the ASIC2a+3 current. The ASIC3‐like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co‐expressed ASIC3‐containing channels.

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life‐threatening Long QT syndrome. These genes encode K+ channel pore‐forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, IKr and IKs, of the human cardiac delayed rectifier current IK. The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates IKs when associated with KCNQ1, whereas it augments the amplitude of an IKr‐like current when co‐expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.

Structure and pharmacology of spider venom neurotoxins.

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution.

Black mamba venom peptides target acid-sensing ion channels to abolish pain

Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Acid-sensing ion channels (ASICs) are generally considered principal players in the pain pathway, including in humans. A snake toxin activating peripheral ASICs in nociceptive neurons has been recently shown to evoke pain. Here we show that a new class of three-finger peptides from another snake, the black mamba, is able to abolish pain through inhibition of ASICs expressed either in central or peripheral neurons. These peptides, which we call mambalgins, are not toxic in mice but show a potent analgesic effect upon central and peripheral injection that can be as strong as morphine. This effect is, however, resistant to naloxone, and mambalgins cause much less tolerance than morphine and no respiratory distress. Pharmacological inhibition by mambalgins combined with the use of knockdown and knockout animals indicates that blockade of heteromeric channels made of ASIC1a and ASIC2a subunits in central neurons and of ASIC1b-containing channels in nociceptors is involved in the analgesic effect of mambalgins. These findings identify new potential therapeutic targets for pain and introduce natural peptides that block them to produce a potent analgesia.

A tarantula peptide against pain via ASIC1a channels and opioid mechanisms

Psalmotoxin 1, a peptide extracted from the South American tarantula Psalmopoeus cambridgei, has very potent analgesic properties against thermal, mechanical, chemical, inflammatory and neuropathic pain in rodents. It exerts its action by blocking acid-sensing ion channel 1a, and this blockade results in an activation of the endogenous enkephalin pathway. The analgesic properties of the peptide are suppressed by antagonists of the μ and δ-opioid receptors and are lost in Penk1−/− mice.

Effects of phrixotoxins on the Kv4 family of potassium channels and implications for the role of Ito1 in cardiac electrogenesis

In the present study, two new peptides, phrixotoxins PaTx1 and PaTx2 (29–31 amino acids), which potently block A‐type potassium currents, have been purified from the venom of the tarantula Phrixotrichus auratus. Phrixotoxins specifically block Kv4.3 and Kv4.2 currents that underlie Ito1, with an 5

Knockout of the ASIC2 channel in mice does not impair cutaneous mechanosensation, visceral mechanonociception and hearing

Mechanosensitive cation channels are thought to be crucial for different aspects of mechanoperception, such as hearing and touch sensation. In the nematode C. elegans, the degenerins MEC‐4 and MEC‐10 are involved in mechanosensation and were proposed to form mechanosensitive cation channels. Mammalian degenerin homologues, the H+‐gated ASIC channels, are expressed in sensory neurones and are therefore interesting candidates for mammalian mechanosensors. We investigated the effect of an ASIC2 gene knockout in mice on hearing and on cutaneous mechanosensation and visceral mechanonociception. However, our data do not support a role of ASIC2 in those facets of mechanoperception.

MIT1, a black mamba toxin with a new and highly potent activity on intestinal contraction

Mamba intestinal toxin (MIT1) isolated from Dendroaspis polylepis venom is a 81 amino acid polypeptide cross‐linked by five disulphide bridges. MIT1 has a very potent action on guinea‐pig intestinal contractility. MIT1 (1 nM) potently contracts longitudinal ileal muscle and distal colon, and this contraction is equivalent to that of 40 mM K+. Conversely MIT1 relaxes proximal colon again as potently as 40 mM K+. The MIT1‐induced effects are antagonised by tetrodotoxin (1 μM) in proximal and distal colon but not in longitudinal ileum. The MIT1‐induced relaxation of the proximal colon is reversibly inhibited by the NO synthase inhibitor L‐NAME (200 μM). 125I‐labelled MIT1 binds with a very high affinity to both ileum and brain membranes (K d=1.3 pM and 0.9 pM, and B max=30 fmol/mg and 26 fmol/mg, respectively). MIT1 is a very highly selective toxin for a receptor present both in the CNS and in the smooth muscle and which might be an as yet unidentified K+ channel.

The bee venom peptide tertiapin underlines the role of IKACh in acetylcholine-induced atrioventricular blocks

Acetylcholine (ACh) is an important neuromodulator of cardiac function that is released upon stimulation of the vagus nerve. Despite numerous reports on activation of IKACh by acetylcholine in cardiomyocytes, it has yet to be demonstrated what role this channel plays in cardiac conduction. We studied the effect of tertiapin, a bee venom peptide blocking IKACh, to evaluate the role of IKACh in Langendorff preparations challenged with ACh. ACh (0.5 μM) reproducibly and reversibly induced complete atrioventricular (AV) blocks in retroperfused guinea‐pig isolated hearts (n=12). Tertiapin (10 to 300 nM) dose‐dependently and reversibly prevented the AV conduction decrements and the complete blocks in unpaced hearts (n=8, P<0.01). Tertiapin dose‐dependently blunted the ACh‐induced negative chronotropic response from an ACh‐induced decrease in heart rate of 39±16% in control conditions to 3±3% after 300 nM tertiapin (P=0.01). These effects were not accompanied by any significant change in QT intervals. Tertiapin blocked IKACh with an IC50 of 30±4 nM with no significant effect on the major currents classically associated with cardiac repolarisation process (IKr, IKs, Ito1, Isus, IK1 or IKATP) or AV conduction (INa and ICa(L)). In summary, tertiapin prevents dose‐dependently ACh‐induced AV blocks in mammalian hearts by inhibiting IKACh.

M-type KCNQ2-KCNQ3 potassium channels are modulated by the KCNE2 subunit.

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K+ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.