Molekulare Mechanismen der Wirkung von Spannungssensor-Toxinen aus Skorpionen und marinen Kegelschnecken auf spannungsgesteuerte Natriumkanäle

Spannungsgesteuerte Natriumkanäle (NaV-Kanäle) sind von entscheidender Bedeutung für die elektrische Signalgebung in erregbaren Zellen von Menschen und Tieren. Es ist deshalb nicht verwunderlich, dass giftige Tiere auch sehr effiziente NaV Kanal-spezifische Toxine entwickelt haben, um ihre Beute schnell zu immobilisieren und zu töten. Viele dieser Toxine haben zudem die Eigenschaft, nur bestimmte NaV Kanäle in ihrer Funktion zu beeinflussen (z.B. nur neuronale NaV Kanäle) und besitzen deshalb auch therapeutisches Potential. In der vorliegenden Dissertation wurden die Wirkungen ausgewählter Skorpion-- und --Toxine sowie und µO-Conotoxine systematisch an heterolog exprimierten NaV-Kanälen aus Säugern mittels der Patch Clamp-Technik untersucht

?O-Conotoxins inhibit NaV channels by interfering with their voltage sensors in domain-2

Journal ArticleThe ?O-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (NaV1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which ?O-conotoxins affect NaV channels. Rat NaV1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily ?-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the NaV channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion ?-toxins. In fact, MrVIA functionally competed with the scorpion ?-toxin Ts1 from Tityus serrulatus, while it did not show competition with ?-GIIIA. Ts1 and ?-GIIIA did not compete either. Thus, similar to scorpion ?-toxins, ?O-conotoxins are voltage-sensor toxins targeting receptor site-4 on NaV channels. They ?block? Na+ flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening

Effect of Conformational Diversity on the Bioactivity of µ-Conotoxin PIIIA Disulfide Isomers

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer’s activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble

Genetic landscape of congenital insensitivity to pain and hereditary sensory and autonomic neuropathies

Congenital insensitivity to pain (CIP) and hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders exclusively or predominantly affecting the sensory and autonomic neurons. Due to the rarity of the diseases and findings based mainly on single case reports or small case series, knowledge about these disorders is limited. Here, we describe the molecular workup of a large international cohort of CIP/HSAN patients including patients from normally under-represented countries. We identify 80 previously unreported pathogenic or likely pathogenic variants in a total of 73 families in the >20 known CIP/HSAN-associated genes. The data expand the spectrum of disease-relevant alterations in CIP/HSAN, including novel variants in previously rarely recognized entities such as ATL3-, FLVCR1- and NGF-associated neuropathies and previously under-recognized mutation types such as larger deletions. In silico predictions, heterologous expression studies, segregation analyses and metabolic tests helped to overcome limitations of current variant classification schemes that often fail to categorize a variant as disease-related or benign. The study sheds light on the genetic causes and disease-relevant changes within individual genes in CIP/HSAN. This is becoming increasingly important with emerging clinical trials investigating subtype or gene-specific treatment strategies

Subtype Specificity of Scorpion ␤-Toxin Tz1 Interaction with Voltage-Gated Sodium Channels Is Determined by the Pore Loop of Domain 3

ABSTRACT Voltage-gated sodium (Na v ) channels are modulated by a variety of specific neurotoxins. Scorpion ␤-toxins affect the voltage-dependence of channel gating: In their presence, Na v channels activate at subthreshold membrane voltages. Previous mutagenesis studies have revealed that the ␤-toxin Css

Combinatorial interaction of scorpion toxins Lqh-2, Lqh-3, and LqhalphaIT with sodium channel receptor sites-3

ABSTRACT Scorpion ␣-toxins Lqh␣IT, Lqh-2, and Lqh-3 are representatives of three groups of ␣-toxins that differ in their preference for insects and mammals. These ␣-insect, antimammalian, and ␣-like toxins bind to voltage-gated sodium channels and slow down channel inactivation. Sodium channel mutagenesis studies using various ␣-toxins have shown that they interact with receptor site 3, which is composed mainly of a short stretch of amino-acid residues between S3 and S4 of domain 4. Variation in this region results in marked differences between various subtypes of sodium channels with respect to their sensitivity to the three Lqh toxins. We incorporated the S3-S4 linker of domain 4 from hNa V 1.2/hNa V 1.1, hNa V 1.3, hNa V 1.6, and hNa V 1.7 channels as well as individual point mutations into the rNa V 1.4 skeletal muscle sodium channel. Our data show that the affinity of Lqh-3 and Lqh␣IT to sodium channels is markedly determined by an aspartate residue (Asp1428 in rNa V 1.4); when mutated to glutamate, as is present in Na V 1.1-1.3 channels, Lqh-3-channel interactions are abolished. The interaction of Lqh-2 and Lqh␣IT, however, is strongly reduced when a lysine residue (Lys1432 in rNa V 1.4) is replaced by threonine (as in hNa V 1.7), whereas this substitution is without effect for Lqh-3. The influence of Lys1432 on Lqh-2 and Lqh␣IT strongly depends on the context of the Asp/Glu site at position 1428, giving rise to a wide variety of toxicological phenotypes by means of a combinatorial mixing and matching of only a few residues in receptor site 3. Voltage-gated sodium (Na V ) channels consist of a large (ϳ260 kDa) pore-forming ␣-subunit, composed of four homologous domains (D1-D4), each with six transmembrane segments (S1-S6) and a hairpin-like pore region between S5 and S6. Because of their structural conservation in vertebrates and invertebrates and their pivotal role in cellular excitability, Na V channels are targeted by a large variety of chemically distinct toxins, many of which do not differentiate among channel subtypes Scorpion toxins affecting Na V channels are 61-to 76-residue polypeptides that comprise two major classes, ␣-and ␤-toxins, according to their mode of action and binding properties to distinct sites (receptor sites-3 and -4, respectively) on Na V channels Despite differences in toxicity and binding properties, all scorpion ␣-toxins bind to receptor site 3, the structural features of which are still elusive, but known to involve the extracellular loops S5-S6 of D1 and D4 Mammalian Na V channels are encoded by a gene family