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.

MLC901 Favors Angiogenesis and Associated Recovery after Ischemic Stroke in Mice.

BACKGROUND: There is increasing evidence that angiogenesis, through new blood vessel formation, results in improved collateral circulation and may impact the long-term recovery of patients. In this study, we first investigated the preventive action of a 5-week pretreatment of MLC901, an herbal extract preparation derived from Chinese medicine, against the deleterious effects of ischemic stroke and its effects on angiogenesis in a model of focal ischemia in mice. METHODS: The stroke model was induced by 60 min of middle cerebral artery occlusion followed by reperfusion. MLC901 was administered in the drinking water of animals (6 g/l) for 5 weeks before ischemia and then during reperfusion. RESULTS: MLC901 treatment increased the survival rate, reduced the cerebral infarct area and attenuated the blood brain barrier leakage as well as the neurologic dysfunction following ischemia and reperfusion. We provide evidence that MLC901 enhances endothelial cell proliferation and angiogenesis by increasing the number of neocortical vessels in the infarcted area. MLC901 regulates the expression of hypoxic inducible factor 1α and its downstream targets such as vascular endothelial growth factor and angiopoietins 1 and 2. This work also shows that erythropoietin is an important player in the enhancement of angiogenesis by MLC901. CONCLUSIONS: These results demonstrate therapeutic properties of MLC901, in addition to those previously described, in stimulating revascularization, neuroprotection and repair of the neurovascular unit after ischemic stroke.

Selective blockade of Cav1.2 (α1C) versus Cav1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2- and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

The TREK-1 potassium channel is involved in both the analgesic and anti-proliferative effects of riluzole in bone cancer pain.

BACKGROUND: The metastasis of tumors into bone tissue typically leads to intractable pain that is both very disabling and particularly difficult to manage. We investigated here whether riluzole could have beneficial effects for the treatment of prostate cancer-induced bone pain and how it could influence the development of bone metastasis. METHODS: We used a bone pain model induced by intratibial injection of human PC3 prostate cancer cells into male SCID mice treated or not with riluzole administered in drinking water. We also used riluzole in vitro to assess its possible effect on PC3 cell viability and functionality, using patch-clamp. RESULTS: Riluzole had a significant preventive effect on both evoked and spontaneous pain involving the TREK-1 potassium channel. Riluzole did not interfere with PC3-induced bone loss or bone remodeling in vivo. It also significantly decreased PC3 cell viability in vitro. The antiproliferative effect of riluzole is correlated with a TREK-1-dependent membrane hyperpolarization in these cells. CONCLUSION: The present data suggest that riluzole could be very useful to manage evoked and spontaneous hypersensitivity in cancer-induced bone pain and has no significant adverse effect on cancer progression.

The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception.

The sensation of cold or heat depends on the activation of specific nerve endings in the skin. This involves heat- and cold-sensitive excitatory transient receptor potential (TRP) channels. However, we show here that the mechano-gated and highly temperature-sensitive potassium channels of the TREK/TRAAK family, which normally work as silencers of the excitatory channels, are also implicated. They are important for the definition of temperature thresholds and temperature ranges in which excitation of nociceptor takes place and for the intensity of excitation when it occurs. They are expressed with thermo-TRP channels in sensory neurons. TRAAK and TREK-1 channels control pain produced by mechanical stimulation and both heat and cold pain perception in mice. Expression of TRAAK alone or in association with TREK-1 controls heat responses of both capsaicin-sensitive and capsaicin-insensitive sensory neurons. Together TREK-1 and TRAAK channels are important regulators of nociceptor activation by cold, particularly in the nociceptor population that is not activated by menthol.

Acid-sensing ion channels in postoperative pain.

Iatrogenic pain consecutive to a large number of surgical procedures has become a growing health concern. The etiology and pathophysiology of postoperative pain are still poorly understood, but hydrogen ions appear to be important in this process. We have investigated the role of peripheral acid-sensing ion channels (ASICs), which form depolarizing channels activated by extracellular protons, in a rat model of postoperative pain (i.e., hindpaw skin/muscle incision). We report high levels of ASIC-type currents (∼ 77%) in sensory neurons innervating the hindpaw muscles, with a prevalence of ASIC3-like currents. The ASIC3 protein is largely expressed in lumbar DRG neurons innervating the plantar muscle, and its mRNA and protein levels are increased by plantar incision 24 h after surgery. Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone. The specific homomeric ASIC1a blocker PcTx1 has no effect on spontaneous flinching, when applied peripherally. Together, these data demonstrate a significant role for peripheral ASIC3-containing channels in postoperative pain.

ASIC3, a sensor of acidic and primary inflammatory pain.

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that approximately 60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (approximately pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Extracellular acidification exerts opposite actions on TREK1 and TREK2 potassium channels via a single conserved histidine residue.

Mechanosensitive K(+) channels TREK1 and TREK2 form a subclass of two P-domain K(+) channels. They are potently activated by polyunsaturated fatty acids and are involved in neuroprotection, anesthesia, and pain perception. Here, we show that acidification of the extracellular medium strongly inhibits TREK1 with an apparent pK near to 7.4 corresponding to the physiological pH. The all-or-none effect of pH variation is steep and is observed within one pH unit. TREK2 is not inhibited but activated by acidification within the same range of pH, despite its close homology with TREK1. A single conserved residue, H126 in TREK1 and H151 in TREK2, is involved in proton sensing. This histidine is located in the M1P1 extracellular loop preceding the first P domain. The differential effect of acidification, that is, activation for TREK2 and inhibition for TREK1, involves other residues located in the P2M4 loop, linking the second P domain and the fourth membrane-spanning segment. Structural modeling of TREK1 and TREK2 and site-directed mutagenesis strongly suggest that attraction or repulsion between the protonated side chain of histidine and closely located negatively or positively charged residues in P2M4 control outer gating of these channels. The differential sensitivity of TREK1 and TREK2 to external pH variations discriminates between these two K(+) channels that otherwise share the same regulations by physical and chemical stimuli, and by hormones and neurotransmitters.

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 mu and delta-opioid receptors and are lost in Penk1-/- mice.

Structural elements for the generation of sustained currents by the acid pain sensor ASIC3.

ASIC3 is an acid-sensing ion channel expressed in sensory neurons, where it participates in acidic and inflammatory pain. In addition to the "classical" transient current, ASIC3 generates a sustained current essential for pain perception. Using chimeras between the ASIC3 and ASIC1a channels we show that the first transmembrane domain (TM1), combined with the N-terminal domain, is the key structural element generating the low pH (<6.5)-evoked sustained current. The TM1 domain also modulates the pH-dependent activation of the fast transient current thus contributing to a constitutive window current, another type of sustained current present near physiological pH. The C-terminal and the TM2 domains negatively regulate both types of sustained current, and the extracellular loop affects its kinetics. These data provide new information to aid understanding the mechanisms of the multifaceted pH gating of ASIC3. Together with the peak current, both components of the sustained current (window and sustained at pH <6.5) allow ASIC3 to adapt its behavior to a wide range of extracellular pH variations by generating transient and/or sustained responses that contribute to nociceptor excitability.

Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype.

Depression is a devastating illness with a lifetime prevalence of up to 20%. The neurotransmitter serotonin or 5-hydroxytryptamine (5-HT) is involved in the pathophysiology of depression and in the effects of antidepressant treatments. However, molecular alterations that underlie the pathology or treatment of depression are still poorly understood. The TREK-1 protein is a background K+ channel regulated by various neurotransmitters including 5-HT. In mice, the deletion of its gene (Kcnk2, also called TREK-1) led to animals with an increased efficacy of 5-HT neurotransmission and a resistance to depression in five different models and a substantially reduced elevation of corticosterone levels under stress. TREK-1-deficient (Kcnk2-/-) mice showed behavior similar to that of naive animals treated with classical antidepressants such as fluoxetine. Our results indicate that alterations in the functioning, regulation or both of the TREK-1 channel may alter mood, and that this particular K+ channel may be a potential target for new antidepressants.

Acid sensing ion channels in dorsal spinal cord neurons.

Acid-sensing ion channels (ASICs) are broadly expressed in the CNS, including the spinal cord. However, very little is known about the properties of ASICs in spinal cord neurons compared with brain. We show here that ASIC1a and ASIC2a are the most abundant ASICs in mouse adult spinal cord and are coexpressed by most neurons throughout all the laminas. ASIC currents in cultured embryonic day 14 mouse dorsal spinal neurons mainly flow through homomeric ASIC1a (34% of neurons) and heteromeric ASIC1a plus 2a channels at a ratio of 2:1 (83% of neurons). ASIC2b only has a minor contribution to these currents. The two channel subtypes show different active pH ranges and different inactivation and reactivation kinetics supporting complementary functional properties. One striking property of native dorsal spinal neuron currents and recombinant currents is the pH dependence of the reactivation process. A light sustained acidosis induces a threefold slow-down of the homomeric ASIC1a (from pH 7.4 to pH 7.3) and heteromeric ASIC1a plus 2a (from pH 7.4 to pH 7.2) current reactivation (T(0.5) increasing from 5.77 to 16.84 s and from 0.98 to 3.2 s, respectively), whereas a larger acidosis to pH 6.6 induces a 32-fold slow-down of the ASIC1a plus 2a current reactivation (T(0.5) values increasing to 31.30 s). The pH dependence of ASIC channel reactivation is likely to modulate neuronal excitability associated with repetitive firing in response to extracellular pH oscillations, which can be induced, for example, by intense synaptic activity of central neurons.

Mtap2 is a constituent of the protein network that regulates twik-related K+ channel expression and trafficking.

Twik-related K+ (TREK) channels produce background currents that regulate cell excitability. In vivo, TREK-1 is involved in neuronal processes including neuroprotection against ischemia, general anesthesia, pain perception, and mood. Recently, we demonstrated that A-kinase anchoring protein AKAP150 binds to a major regulatory domain of TREK-1, promoting drastic changes in channel regulation by polyunsaturated fatty acids, pH, and stretch, and by G-protein-coupled receptors to neurotransmitters and hormones. Here, we show that the microtubule-associated protein Mtap2 is another constituent of native TREK channels in the brain. Mtap2 binding to TREK-1 and TREK-2 does not affect directly channel properties but enhances channel surface expression and current density. This effect relies on Mtap2 binding to microtubules. Mtap2 and AKAP150 interacting sites in TREK-1 are distinct and both proteins can dock simultaneously. Their effects on TREK-1 surface expression and activation are cumulative. In neurons, the three proteins are simultaneously detected in postsynaptic dense bodies. AKAP150 and Mtap2 put TREK channels at the center of a complex protein network that finely tunes channel trafficking, addressing, and regulation.

Sea anemone toxins affecting potassium channels.

The great diversity of K(+) channels and their wide distribution in many tissues are associated with important functions in cardiac and neuronal excitability that are now better understood thanks to the discovery of animal toxins. During the past few decades, sea anemones have provided a variety of toxins acting on voltage-sensitive sodium and, more recently, potassium channels. Currently there are three major structural groups of sea anemone K(+) channel (SAK) toxins that have been characterized. Radioligand binding and electrophysiological experiments revealed that each group contains peptides displaying selective activities for different subfamilies of K(+) channels. Short (35-37 amino acids) peptides in the group I display pore blocking effects on Kv1 channels. Molecular interactions of SAK-I toxins, important for activity and binding on Kv1 channels, implicate a spot of three conserved amino acid residues (Ser, Lys, Tyr) surrounded by other less conserved residues. Long (58-59 amino acids) SAK-II peptides display both enzymatic and K(+) channel inhibitory activities. Medium size (42-43 amino acid) SAK-III peptides are gating modifiers which interact either with cardiac HERG or Kv3 channels by altering their voltage-dependent properties. SAK-III toxins bind to the S3C region in the outer vestibule of Kv channels. Sea anemones have proven to be a rich source of pharmacological tools, and some of the SAK toxins are now useful drugs for the diagnosis and treatment of autoimmune diseases.

Polyunsaturated fatty acids are cerebral vasodilators via the TREK-1 potassium channel.

Vessel occlusion is the most frequent cause for impairment of local blood flow within the brain resulting in neuronal damage and is a leading cause of disability and death worldwide. Polyunsaturated fatty acids and especially alpha-linolenic acid improve brain resistance against cerebral ischemia. The purpose of the present study was to evaluate the effects of polyunsaturated fatty acids and particularly alpha-linolenic acid on the cerebral blood flow and on the tone of vessels that regulate brain perfusion. alpha-Linolenic acid injections increased cerebral blood flow and induced vasodilation of the basilar artery but not of the carotid artery. The saturated fatty acid palmitic acid did not produce vasodilation. This suggested that the target of the polyunsaturated fatty acids effect was the TREK-1 potassium channel. We demonstrate the presence of this channel in basilar but not in carotid arteries. We show that vasodilations induced by the polyunsaturated fatty acid in the basilar artery as well as the laser-Doppler flow increase are abolished in TREK-1(-/-) mice. Altogether these data indicate that TREK-1 activation elicits a robust dilation that probably accounts for the increase of cerebral blood flow induced by polyunsaturated fatty acids such as alpha-linolenic acid or docosahexanoic acid. They suggest that the selective expression and activation of TREK-1 in brain collaterals could play a significant role in the protective mechanisms of polyunsaturated fatty acids against stroke by providing residual circulation during ischemia.

Chemical Synthesis, Proper Folding, Nav Channel Selectivity Profile and Analgesic Properties of the Spider Peptide Phlotoxin 1.

Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (Phlogius species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Nav) channel Nav1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Nav1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Nav1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Nav1.7 involvement in cellular excitability and pain.

Targeting the TREK-1 potassium channel via riluzole to eliminate the neuropathic and depressive-like effects of oxaliplatin.

Neurotoxicity remains the most common adverse effect of oxaliplatin, limiting its clinical use. In the present study, we developed a mouse model of chronic oxaliplatin-induced neuropathy, which mimics both sensory and motor deficits observed in patients, in a clinically relevant time course. Repeated oxaliplatin administration in mice induced both cephalic and extracephalic long lasting mechanical and cold hypersensitivity after the first injection as well as delayed sensorimotor deficits and a depression-like phenotype. Using this model, we report that riluzole prevents both sensory and motor deficits induced by oxaliplatin as well as the depression-like phenotype induced by cumulative chemotherapeutic drug doses. All the beneficial effects are due to riluzole action on the TREK-1 potassium channel, which plays a central role in its therapeutic action. Riluzole has no negative effect on oxaliplatin antiproliferative capacity in human colorectal cancer cells and on its anticancer effect in a mouse model of colorectal cancer. Moreover, riluzole decreases human colorectal cancer cell line viability in vitro and inhibits polyp development in vivo. The present data in mice may support the need to clinically test riluzole in oxaliplatin-treated cancer patients and state for the important role of the TREK-1 channel in pain perception.

Silencing acid-sensing ion channel 1a alters cone-mediated retinal function.

The action of extracellular protons on retinal activity and phototransduction occurs through pH-sensitive elements, mainly membrane conductances present on the different cell types of the outer and inner nuclear layers and of the ganglion cell layer. Acid-sensing ion channels (ASICs) are depolarizing conductances that are directly activated by protons. We investigated the participation of ASIC1a, a particular isoform of ASICs, in retinal physiology in vivo using electroretinogram measurements. In situ hybridization and immunohistochemistry localized ASIC1a in the outer and inner nuclear layers (cone photoreceptors, horizontal cells, some amacrine and bipolar cells) and in the ganglion cell layer. Both the in vivo knockdown of ASIC1a by antisense oligonucleotides and the in vivo blocking of its activity by PcTx1, a specific venom peptide, were able to decrease significantly and reversibly the photopic a- and b-waves and oscillatory potentials. Our study indicates that ASIC1a is an important channel in normal retinal activity. Being present in the inner segments of cones and inner nuclear layer cells, and mainly at synaptic cleft levels, it could participate in gain adaptation to ambient light of the cone pathway, facilitating cone hyperpolarization in brightness and modulating synaptic transmission of the light-induced visual signal.