Frank Bosmans

Deconstructing voltage sensor function and pharmacology in sodium channels

Voltage-activated sodium (Nav) channels are crucial for the generation and propagation of nerve impulses, and as such are widely targeted by toxins and drugs. The four voltage sensors in Nav channels have distinct amino acid sequences, raising fundamental questions about their relative contributions to the function and pharmacology of the channel. Here we use four-fold symmetric voltage-activated potassium (Kv) channels as reporters to examine the contributions of individual S3b–S4 paddle motifs within Nav channel voltage sensors to the kinetics of voltage sensor activation and to forming toxin receptors. Our results uncover binding sites for toxins from tarantula and scorpion venom on each of the four paddle motifs in Nav channels, and reveal how paddle-specific interactions can be used to reshape Nav channel activity. One paddle motif is unique in that it slows voltage sensor activation, and toxins selectively targeting this motif impede Nav channel inactivation. This reporter approach and the principles that emerge will be useful in developing new drugs for treating pain and Nav channelopathies.

Targeting voltage sensors in sodium channels with spider toxins

Voltage-activated sodium (Nav) channels are essential in generating and propagating nerve impulses, placing them amongst the most widely targeted ion channels by toxins from venomous organisms. An increasing number of spider toxins have been shown to interfere with the voltage-driven activation process of mammalian Nav channels, possibly by interacting with one or more of their voltage sensors. This review focuses on our existing knowledge of the mechanism by which spider toxins affect Nav channel gating and the possible applications of these toxins in the drug discovery process.

Interactions between lipids and voltage sensor paddles detected with tarantula toxins

Voltage-activated ion channels open and close in response to changes in voltage, a property that is essential for generating nerve impulses. Studies on voltage-activated potassium (Kv) channels show that voltage-sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1–S4 voltage-sensing domains and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters voltage-sensor activation in an S1–S4 voltage-sensing protein lacking an associated pore domain, and that the S3b–S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to voltage-sensing domains and demonstrate that the pharmacological sensitivities of voltage-activated ion channels are influenced by the surrounding lipid membrane.

Adaptive Evolution of Scorpion Sodium Channel Toxins

Gene duplication followed by positive Darwinian selection is an important evolutionary event at the molecular level, by which a gene can gain new functions. Such an event might have occurred in the evolution of scorpion sodium channel toxin genes (α- and β-groups). To test this hypothesis, a robust statistical method from Yang and co-workers based on the estimation of the nonsynonymous-to-synonymous rate ratio (ω = dN/dS) was performed. The results provide clear statistical evidence for adaptive molecular evolution of scorpion α- and β-toxin genes. A good match between the positively selected sites (evolutionary epitopes) and the putative bioactive surface (functional epitopes) indicates that these sites are most likely involved in functional recognition of sodium channels. Our results also shed light on the importance of the B-loop in the functional diversification of scorpion α- and β-toxins.

Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

Animal Peptides Targeting Voltage-Activated Sodium Channels

Throughout millions of years of evolution, nature has supplied various organisms with a massive arsenal of venoms to defend themselves against predators or to hunt prey. These venoms are rich cocktails of diverse bioactive compounds with divergent functions, extremely effective in immobilizing or killing the recipient. In fact, venom peptides from various animals have been shown to specifically act on ion channels and other cellular receptors, and impair their normal functioning. Because of their key role in the initiation and propagation of electrical signals in excitable tissue, it is not very surprising that several isoforms of voltage-activated sodium channels are specifically targeted by many of these venom peptides. Therefore, these peptide toxins provide tremendous opportunities to design drugs with a higher efficacy and fewer undesirable side effects. This review puts venom peptides from spiders, scorpions and cone snails that target voltage-activated sodium channels in the spotlight, and addresses their potential therapeutical applications.

Function and solution structure of hainantoxin-I, a novel insect sodium channel inhibitor from the Chinese bird spider Selenocosmia hainana

Hainantoxin‐I is a novel peptide toxin, purified from the venom of the Chinese bird spider Selenocosmia hainana (=Ornithoctonus hainana). It includes 33 amino acid residues with a disulfide linkage of I–IV, II–V and III–VI, assigned by partial reduction and sequence analysis. Under two‐electrode voltage‐clamp conditions, hainantoxin‐I can block rNav1.2/β1 and the insect sodium channel para/tipE expressed in Xenopus laevis oocytes with IC50 values of 68±6 μM and 4.3±0.3 μM respectively. The three‐dimensional solution structure of hainantoxin‐I belongs to the inhibitor cystine knot structural family determined by two‐dimensional 1H nuclear magnetic resonance techniques. Structural comparison of hainantoxin‐I with those of other toxins suggests that the combination of the charged residues and a vicinal hydrophobic patch should be responsible for ligand binding. This is the first report of an insect sodium channel blocker from spider venom and it provides useful information for the structure–function relationship studies of insect sodium channels.

From foe to friend: using animal toxins to investigate ion channel function.

Ion channels are vital contributors to cellular communication in a wide range of organisms, a distinct feature that renders this ubiquitous family of membrane-spanning proteins a prime target for toxins found in animal venom. For many years, the unique properties of these naturally occurring molecules have enabled researchers to probe the structural and functional features of ion channels and to define their physiological roles in normal and diseased tissues. To illustrate their considerable impact on the ion channel field, this review will highlight fundamental insights into toxin-channel interactions and recently developed toxin screening methods and practical applications of engineered toxins.

The hitchhiker’s guide to the voltage-gated sodium channel galaxy

Eukaryotic voltage-gated sodium (Nav) channels contribute to the rising phase of action potentials and served as an early muse for biophysicists laying the foundation for our current understanding of electrical signaling. Given their central role in electrical excitability, it is not surprising that (a) inherited mutations in genes encoding for Nav channels and their accessory subunits have been linked to excitability disorders in brain, muscle, and heart; and (b) Nav channels are targeted by various drugs and naturally occurring toxins. Although the overall architecture and behavior of these channels are likely to be similar to the more well-studied voltage-gated potassium channels, eukaryotic Nav channels lack structural and functional symmetry, a notable difference that has implications for gating and selectivity. Activation of voltage-sensing modules of the first three domains in Nav channels is sufficient to open the channel pore, whereas movement of the domain IV voltage sensor is correlated with inactivation. Also, structure–function studies of eukaryotic Nav channels show that a set of amino acids in the selectivity filter, referred to as DEKA locus, is essential for Na+ selectivity. Structures of prokaryotic Nav channels have also shed new light on mechanisms of drug block. These structures exhibit lateral fenestrations that are large enough to allow drugs or lipophilic molecules to gain access into the inner vestibule, suggesting that this might be the passage for drug entry into a closed channel. In this Review, we will synthesize our current understanding of Nav channel gating mechanisms, ion selectivity and permeation, and modulation by therapeutics and toxins in light of the new structures of the prokaryotic Nav channels that, for the time being, serve as structural models of their eukaryotic counterparts.

Crystallographic insights into sodium-channel modulation by the β4 subunit

Significance Voltage-gated sodium (Nav) channels are members of a large complex that plays a crucial role in rapid electrical signaling throughout the human body. As prominent members of this complex, β-subunits modify Nav channel function and cause debilitating disorders when mutated. Collectively, the functional and crystallographic results reported in this work uncover intricate interactions of these elements within the Nav-channel signaling complex and establish a key role for β-subunits in shaping Nav1.2 pharmacology. An important concept emerging from our results is that β-subunits provide exciting opportunities for designing new therapeutic strategies to correct their abnormal behaviors. Voltage-gated sodium (Nav) channels are embedded in a multicomponent membrane signaling complex that plays a crucial role in cellular excitability. Although the mechanism remains unclear, β-subunits modify Nav channel function and cause debilitating disorders when mutated. While investigating whether β-subunits also influence ligand interactions, we found that β4 dramatically alters toxin binding to Nav1.2. To explore these observations further, we solved the crystal structure of the extracellular β4 domain and identified 58Cys as an exposed residue that, when mutated, eliminates the influence of β4 on toxin pharmacology. Moreover, our results suggest the presence of a docking site that is maintained by a cysteine bridge buried within the hydrophobic core of β4. Disrupting this bridge by introducing a β1 mutation implicated in epilepsy repositions the 58Cys-containing loop and disrupts β4 modulation of Nav1.2. Overall, the principles emerging from this work (i) help explain tissue-dependent variations in Nav channel pharmacology; (ii) enable the mechanistic interpretation of β-subunit–related disorders; and (iii) provide insights in designing molecules capable of correcting aberrant β-subunit behavior.