Enrico Leipold

A de novo gain-of-function mutation in SCN11A causes loss of pain perception

The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.

Molecular interaction of δ-conotoxins with voltage-gated sodium channels

Various neurotoxic peptides modulate voltage‐gated sodium (NaV) channels and thereby affect cellular excitability. δ‐Conotoxins from predatory cone snails slow down inactivation of NaV channels, but their interaction site and mechanism of channel modulation are unknown. Here, we show that δ‐conotoxin SVIE from Conus striatus interacts with a conserved hydrophobic triad (YFV) in the domain‐4 voltage sensor of NaV channels. This site overlaps with that of the scorpion α‐toxin Lqh‐2, but not with the α‐like toxin Lqh‐3 site. δ‐SVIE functionally competes with Lqh‐2, but exhibits strong cooperativity with Lqh‐3, presumably by synergistically trapping the voltage sensor in its “on” position.

Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in mammalian cells was studied by applying the Met-preferring oxidant chloramine-T or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile–Phe–Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of Met1470 in the putative receptor of the inactivation lid also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues in intracellular linkers connecting the membrane-spanning segments (442, 1139, 1154, 1316, 1469) were of minor importance. The results of mutagenesis, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7), and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion.

The μO-conotoxin MrVIA inhibits voltage-gated sodium channels by associating with domain-3

Several families of peptide toxins from cone snails affect voltage‐gated sodium (NaV) channels: μ‐conotoxins block the pore, δ‐conotoxins inhibit channel inactivation, and μO‐conotoxins inhibit NaV channels by an unknown mechanism. The only currently known μO‐conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (NaV1.4) and brain (NaV1.2) sodium channels in mammalian cells. A systematic domain‐swapping strategy identified the C‐terminal pore loop of domain‐3 as the major determinant for NaV1.4 being more potently blocked than NaV1.2 channels. μO‐conotoxins therefore show an interaction pattern with NaV channels that is clearly different from the related μ‐ and δ‐conotoxins, indicative of a distinct molecular mechanism of channel inhibition.

Differential sensitivity of sodium channels from the central and peripheral nervous system to the scorpion toxins Lqh-2 and Lqh-3.

The scorpion α‐toxins Lqh‐2 and Lqh‐3, isolated from the venom of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus, were previously shown to be very potent in removing fast inactivation of rat skeletal muscle sodium channels ( Chen et al., 2000 ). Here, we show that tetrodotoxin‐sensitive neuronal channels NaV1.2 and NaV1.7, which are mainly expressed in mammalian central and peripheral nervous systems, respectively, are differentially sensitive to these two toxins. rNaV1.2 and hNaV1.7 channels were studied with patch‐clamp methods upon expression in mammalian cells. While Lqh‐3 was about 100‐times more potent in removing inactivation in hNaV1.7 channels compared with rNaV1.2, Lqh‐2 was about 20‐times more active in the other direction. Site‐directed mutagenesis showed that the differences in the putative binding sites for these toxins, the S3‐4 linkers of domain 4, are of major importance for Lqh‐3, but not for Lqh‐2.

Micro-structured smart hydrogels with enhanced protein loading and release efficiency.

A series of temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm) hydrogels with highly porous microstructures were successfully prepared by using hydrophobic polydimethylsiloxane (PDMS) and sodium dodecyl sulfate as liquid template and stabilizer, respectively. These newly prepared hydrogels possess highly porous structures. In contrast to the conventional PNIPAAm hydrogel, the swelling ratios of the porous gels at room temperature were higher, and their response rates were significantly faster as the temperature was raised above the lower critical solution temperature. For example, the novel hydrogel prepared with 40% PDMS template lost over 95% water within 5 min, while the conventional PNIPAAm gel only lost approximately 14% water in the same time. The improved properties are achieved due to the presence of liquid PDMS templates in the reaction solutions, which lead to the formation of porous structures during the polymerization/crosslinking. Lysozyme and bovine serum albumin (BSA) as protein models were for the first time loaded into these micro-structured smart hydrogels through a physical absorption method. The experimental results show that the loading efficiency of BSA with a higher molecular weight is lower than that of lysozyme due to the size exclusion effect, and the loading efficiencies of both proteins in the porous hydrogel are much higher than those in the conventional PNIPAAm hydrogel. For example, the loading efficiency of BSA in porous hydrogel is 0.114, approximately 200% higher than that in conventional hydrogel (0.035). Both lysozyme and BSA were completely released from the porous hydrogel at 22 degrees C. Furthermore, the release kinetics of the proteins from the porous hydrogel could be modulated by tuning the environmental temperature. These newly prepared porous materials provide an avenue to increase the loading efficiency and to control the release patterns of macromolecular drugs from hydrogels, and show great promise for application in protein or gene delivery.

Conotoxins of the O-superfamily affecting voltage-gated sodium channels

Abstract.The venoms of predatory cone snails harbor a rich repertoire of peptide toxins that are valuable research tools, but recently have also proven to be useful drugs. Among the conotoxins with several disulfide bridges, the O-superfamily toxins are characterized by a conserved cysteine knot pattern: C-C-CC-C-C. While ω-conotoxins and κ-conotoxins block Ca2+ and K+ channels, respectively, the closely related δ- and μO-conotoxins affect voltage-gated Na+ channels (Nav channels). δ-conotoxins mainly remove the fast inactivation of Nav channels and, thus, functionally resemble long-chain scorpion α-toxins. μO-conotoxins are functionally similar to μ-conotoxins, since they inhibit the ion flow through Nav channels. Recent results from functional and structural assays have gained insight into the underlying molecular mechanisms. Both types of toxins are voltage-sensor toxins interfering with the voltage-sensor elements of Nav channels.

Cold-aggravated pain in humans caused by a hyperactive NaV1.9 channel mutant

Gain-of-function mutations in the human SCN11A-encoded voltage-gated Na+ channel NaV1.9 cause severe pain disorders ranging from neuropathic pain to congenital pain insensitivity. However, the entire spectrum of the NaV1.9 diseases has yet to be defined. Applying whole-exome sequencing we here identify a missense change (p.V1184A) in NaV1.9, which leads to cold-aggravated peripheral pain in humans. Electrophysiological analysis reveals that p.V1184A shifts the voltage dependence of channel opening to hyperpolarized potentials thereby conferring gain-of-function characteristics to NaV1.9. Mutated channels diminish the resting membrane potential of mouse primary sensory neurons and cause cold-resistant hyperexcitability of nociceptors, suggesting a mechanistic basis for the temperature dependence of the pain phenotype. On the basis of direct comparison of the mutations linked to either cold-aggravated pain or pain insensitivity, we propose a model in which the physiological consequence of a mutation, that is, augmented versus absent pain, is critically dependent on the type of NaV1.9 hyperactivity.

A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors.

The µ‐conopeptide family is defined by its ability to block voltage‐gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ‐conopeptide (µ‐CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant.

Structurally Diverse μ‐Conotoxin PIIIA Isomers Block Sodium Channel NaV1.4

Certain VGSC subtypes (NaV1.3, 1.7, 1.8, and 1.9) are expressed in the peripheral nervous system and mediate the transmission of signals leading to the sensation of different kinds of pain, such as nociception (NaV1.8), acute inflammatory (NaV1.7), and neuropathic (NaV1.3) pain. [2] Therefore, VGSCs are potential targets for novel analgesics, ideally those with strong channel specificity. Among sodium channel antagonists, m- and mO-conotoxins from the venoms of marine cone snails have attracted considerable attention because of their analgesic potency. [2a, 3] m-Conotoxins are 14to 26-mer peptides with six cysteine residues (Supporting Information, Table S1). [4] They inhibit muscle and/or neuronal VGSCs by occluding the ion channel pore. [5] A specific cysteine framework, that is, CCXnCXnCXnCC, confers conformational restriction to their three-dimensional structure upon formation of three disulfide bonds. It is generally accepted that the native fold of the toxins carries the disulfide connectivities Cys1–Cys4, Cys2–Cys5, and Cys3–Cys6 (numbered in the order of occurrence in the amino acid sequence). [3, 5b] However, three-dimensional structures are available only for a limited subset of m-conotoxins, that is, PIIIA, [6]