Structures of muO-conotoxins from Conus marmoreus. I nhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons

The microO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxin-resistant (TTX-R) Na(+) current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxin-sensitive Na(+) currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the microO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small beta-sheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone "loops" between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known delta- and omega-conotoxins, which along with the microO-conotoxins are members of the O superfamily. Loop 2 of omega-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels, and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels.     

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

Several families of peptide toxins from cone snails affect voltage-gated sodium (Na(V)) channels: mu-conotoxins block the pore, delta-conotoxins inhibit channel inactivation, and muO-conotoxins inhibit Na(V) channels by an unknown mechanism. The only currently known muO-conotoxins MrVIA and MrVIB from Conus marmoreus were applied to cloned rat skeletal muscle (Na(V)1.4) and brain (Na(V)1.2) sodium channels in mammalian cells. A systematic domain-swapping strategy identified the C-terminal pore loop of domain-3 as the major determinant for Na(V)1.4 being more potently blocked than Na(V)1.2 channels. muO-conotoxins therefore show an interaction pattern with Na(V) channels that is clearly different from the related mu- and delta-conotoxins, indicative of a distinct molecular mechanism of channel inhibition.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Arg20突变对敬钊缨毛蛛毒素-V钠通道活性的影响

敬钊缨毛蛛毒素-V(Jingzhaotoxin-V, JZTX-V)是从敬钊缨毛蛛粗毒中纯化到的一种新型河豚毒素不敏感型钠通道抑制剂, 为了深入研究该毒素的结构与功能关系, 应用芴甲氧羰基(Fmoc)固相多肽化学合成方法合成了用丙氨酸(Ala)替代JZTX-V第20位精氨酸残基的突变体R20A-JZTX-V, 合成线性多肽经反相高效液相色谱分离纯化后进行谷胱甘肽氧化复性。复性产物分别用基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS)进行分子量的鉴定, 用膜片钳电生理方法进行电压门控钠通道抑制活性分析。研究结果表明, Arg20被Ala取代后, R20A-JZTX-V对大鼠背根神经节细胞(DRG)膜上表达的河豚毒素敏感型(TTX-S)钠通道的抑制活性与天然JZTX-V相当, 提示Arg20与JZTX-V对TTX-S钠通道的抑制活性无关或关系不大; 而R20A-JZTX-V对TTX-R钠通道的抑制活性却比天然JZTX-V下降了约18.3倍, 说明Arg20是与JZTX-V对河豚毒素不敏感型(TTX-R)钠通道抑制活性相关的关键活性残基之一, 推测R20A-JZTX-V活性降低的原因是用Ala替代Arg20后改变了JZTX-V与TTX-R型钠通道的作用位点。     

The cross channel activities of spider neurotoxin huwentoxin-I on rat dorsal root ganglion neurons

In this paper, we investigated the action of huwentoxin-I (HWTX-I) purified from the venom of the Chinese bird spider Ornithoctonus huwena on Ca(2+), Na(+) channels of adult rat dorsal root ganglion (DRG) neurons. The results showed that huwentoxin-I could reduce the peak currents of N-type Ca(2+) channels (IC(50) approximately 100 nM) and TTX-S Na(+) channels (IC(50) approximately 55 nM), whereas no effect was detected on TTX-R Na(+) channels. The comparative studies indicated that the selectivity of HWTX-I on Ca(2+) channels was higher that of MVIIA and approximately the same as that of GVIA. HWTX-I is the first discovered toxin with the cross channel activities from the spider O. huwena venom similar to micro O-conotoxins MrVIA and MrVIB.     

A naturally-occurring carboxyl-terminally truncated α-scorpion toxin is a blocker of sodium channels

α-Scorpion toxins constitute a multigene family of evolutionarily conserved venom peptides that inhibit sodium channel inactivation and increase its peak current. Here, we describe the characterization of a new α-scorpion toxin gene expressed in the venom gland of Mesobuthus eupeus that encodes a carboxyl-terminally truncated product of 38 residues (named MeuNaTxα(NT)-1). Synthetic MeuNaTxα(NT)-1 was oxidized to form two disulfide bridges in an alkaline environment and the refolded peptide exhibits different structure and function from the classical α-scorpion toxin. MeuNaTxα(NT)-1 blocks sodium channels on rat dorsal root ganglia (DRG) neurons without impact on the inactivation of the channels. This work provides a clue for evolution-guided design of channel blockers for therapeutic aims.     

Competition for Binding Between Veratridine and KIFMK: An Open Channel Blocking Peptide of the RIIA Sodium Channel

Veratridine, an alkaloid isolated from the rhizome of V. album, binds and slows the inactivation of the brain sodium channels. The synthetic pentapeptide KIFMK causes a voltage- and use-dependent open-channel block of the RIIA (rat brain type IIA) sodium channel (Eaholtz, Scheuer & Catterall, 1994). Our studies on the RIIA sodium channel expressed in CHO cells reveal that the fraction of veratridine modified sodium channels decreases linearly with increasing KIFMK concentration. However, the time constant for dissociation of veratridine from the channel remains unchanged in the presence of a high concentration of KIFMK, as opposed to that in the presence of QX314 where the dissociation appears to be more complex. These data are consistent with mutually exclusive binding of the open channel blocking peptide and veratridine to the brain sodium channel. Received: 19 November 1996/Revised: 31 July 1997     

mu-conotoxin GIIIA, a peptide ligand for muscle sodium channels: chemical synthesis, radiolabeling, and receptor characterization

The peptide conotoxin GIIIA from Conus geographus L. venom, which specifically blocks sodium channels in muscle, has been synthesized by a solid-phase method. The three disulfide bridges were formed by air oxidation. After HPLC purification, the synthetic product was shown to be identical with the native conotoxin GIIIA from Conus geographus. A high specific activity, 125I derivative of mu-conotoxin was prepared and used for binding assays to the Na channel from Electrophorus electric organ. Specific binding could be abolished by competition with tetrodotoxin. The radiolabeled toxin was specifically cross-linked to the Na channel. These studies demonstrate that mu-conotoxin GIIIA can be used to define the guanidinium toxin binding site and will be a useful ligand for understanding functionally important differences between Na channel subtypes.     

Synthesis and characterization of delta-atracotoxin-Ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

Delta-atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. (1)H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.     

Mu-conotoxin SmIIIA,a potent inhibitor of tetrodotoxin-resistant sodium channels in amphibian sympathetic and sensory neurons

Mu-conotoxins are a family of peptides from the venoms of predatory cone snails. Previously characterized mu-conotoxins preferentially block skeletal muscle voltage-gated sodium channels. We report here the discovery (via cloning), synthesis, and electrophysiological characterization of a new peptide in this family, mu-conotoxin SmIIIA from Conus stercusmuscarum. Although mu-conotoxin SmIIIA shares several biochemical characteristics with other mu-conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has distinctive features such as the absence of hydroxyproline. In voltage-clamped dissociated neurons from frog sympathetic and dorsal root ganglia, the peptide inhibited the majority of tetrodotoxin-resistant sodium currents irreversibly; in contrast, tetrodotoxin-sensitive sodium currents were largely unaffected by the peptide. We believe that mu-conotoxin SmIIIA is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be discovered. Thus, the peptide provides a new and potentially useful tool to investigate the functional roles of tetrodotoxin-resistant voltage-gated sodium channels, including those that are found in sensory nerves that convey nociceptive information.     

A novel conus peptide ligand for K+ channels

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.     

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.     

Secondary structure of an isolated P-region from the voltage-gated sodium channel: a molecular modelling/dynamics study

Conformational studies of synthetic peptides corresponding to the pore-forming regions of voltage-gated sodium channels show a high tendency for beta-sheet conformation when interacting with lipid vesicles, as revealed by circular dichroism and infrared spectroscopy. These observations have guided our choice of possible molecular models for the P-region peptide of domain II of voltage-gated sodium channels: three alternative beta-hairpins, with differing turn assignments, or an alpha-helical hairpin. After generation of models by distance geometry-based methods, molecular dynamics (MD) simulations were run. in the absence of explicit solvent molecules but employing three different dielectric constants, to explore possible conformational preferences. The simulations in the different dielectric environments suggest that a 4-residue turn with the sequence LCGE yields more stable beta-hairpins. The MD results suggest that the SS1 part of the peptide may be more stable as an alpha-helix, whereas the SS2 part tends to adopt a beta-conformation.     

Generation of polyclonal antibody against mu-conotoxin GIIIA using an immunogen of [Cys(5)]mu-conotoxin GIIIA site-specifically conjugated with bovine serum albumin.

mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins.     

Bay K 8644-like contractile effects of a peptide isolated from spontaneously hypertensive rats

The in vitro contractile effect of a peptide recently isolated from the blood of spontaneously hypertensive rats was assessed on rat aortic rings. Preincubation of aortic rings with the peptide had no effect on resting tension but significantly enhanced K+ or norepinephrine-induced contractile responses. Contractile effects were abolished by removal of extracellular calcium or by additions of the calcium channel antagonists, verapamil and nifedipine. The antagonism of peptide enhancement of contraction by verapamil was noncompetitive, whereas nifedipine blockade was competitive in nature. Moreover, preincubation of aortic rings with the peptide attenuated the contractile response to Bay K 8644, a newly described synthetic calcium channel agonist. We suggest that this peptide has similar effects to Bay K 8644 and may act as an endogenous modulator of voltage-dependent calcium channels.     

Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor--a model study.

The inhibitory glycine receptor (GlyR) of rat spinal cord contains an intrinsic transmembrane channel mediating agonist-gated anion flux. Here, synthetic peptides modelled after the predicted transmembrane domains M2 and M4 of its ligand-binding subunit were incorporated into lipid vesicle membranes and black lipid bilayers to analyze their channel forming capabilities. Both types of peptides prohibited the establishment of, or dissipated, preexisting transmembrane potentials in the vesicle system. Incorporation of peptide M2 into the black lipid bilayer elicited randomly gated single channel events with various conductance states and life-times. Peptide M4 increased the conductance of the bilayer without producing single channels. Exchange of the terminal arginine residues of peptide M2 by glutamate resulted in a significant shift towards cation selectivity of the respective channels as compared to peptide M2. In conclusion, the peptide channels observed differed significantly from native GlyR in both conductivity and ion-selectivity indicating that individual synthetic transmembrane segments are not sufficient to mimic a channel protein composed of subunits with multiple transmembrane segments.     

Active site of mu-conotoxin GIIIA, a peptide blocker of muscle sodium channels.

The amino acid sequence of mu-conotoxin GIIIA (otherwise called geographutoxin I), a peptide having 22 amino acid residues with three disulfide bridges, was modified by replacing each residue with Ala or Lys to elucidate its active center for blocking sodium channels of skeletal muscle. NMR and CD spectra were virtually identical between native and modified toxins, indicating the similarity of their conformation including disulfide bridges. The inhibitory effect of these modified peptides on twitch contractions of the rat diaphragm showed that Arg at the 13th position and the basicity of the molecule are crucial for the biological action. The segment Lys11-Asp12-Arg13 has been reported to be flexible (Lancelin, J.-M., Kohda, D., Tate, S., Yanagawa, Y., Abe, T., Satake, M., and Inagaki, F. (1991) Biochemistry, in press), and this may represent a clue for the subtle fit of Arg13 to the specific site of sodium channels. Since known ligands to sodium channels, such as tetrodotoxin, anthopleulin-A, etc., contain guanidino groups as a putative binding moiety, Arg may be a general residue for peptide toxins to interact with the receptor site on sodium channels.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.