Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (K_i) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a K_i of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with K_i of 1–4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys₂₁ and Tyr₂₂, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg₂₃, indicating that Lys₂₁ is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.Venomous animals produce neurotoxic peptides for defense and to capture prey. With potencies in the nanomolar range, the peptides act by modulating the function of target receptors. Toxins isolated from venoms have been used to identify and purify ion channels, to clarify their roles in physiology, to elucidate the structural basis for their function, and, recently, to diagnose and treat disease. Given their utility, it is frustrating that natural toxins cross-react with related receptors (or have no known target) and that most receptors lack a specific, high-affinity toxin. This state of affairs is easily understood; the small amounts of toxins isolated from natural sources makes target identification a challenge and their purpose in the wild does not favor target specificity. Here, we advance our approach to overcoming these problems, that is, creation of expression libraries of toxins allowing cloning based on target binding (1), by seeking a specific, high-affinity ligand for an orphan channel receptor.Our strategy is to start with a known toxin and to design a phage-display library using the genetic database of its predicted homologs, in native and combinatorial fashion, so the encoded peptides share the same structural scaffold. As a proof of concept, we previously addressed a case of inadequate target discrimination by known natural toxins using a library of ∼11,200 peptides designed to share the fold in α-KTx scorpion toxins and a specific ligand for the human voltage-gated potassium channel Kv1.3 was isolated (1). Moka1, composed of domains from three scorpion species, blocks Kv1.3 with nanomolar affinity, allowing it to suppress T-cell-mediated immune responses, and is without unwanted side effects on gastrointestinal motility seen with natural toxins because it does not cross-inhibit Kv1.1 and Kv1.2. Supporting the design premise that encoded peptides are expressed, correctly folded, and accessible on the phage surface in a manner permissive of sorting based on target binding, the determined 3D structure of Moka1 revealed it to be constructed on an α-KTx scaffold. Here, we sought to extend our strategy by testing another scaffold and creation of a library sufficiently large to achieve isolation of peptides specific for a target with no known ligand.KcsA is a prokaryotic channel with high potassium conductance and selectivity (2). The first potassium channel visualized at high resolution (3), KcsA has a single ion conduction pathway on the central axis of symmetry formed by four identical subunits, each with two transmembrane segments and a reentrant pore-forming loop (TM1-P-TM2). The 3D structure of KcsA confirmed explanations for selective ion permeation and conduction pathway gating deduced in the period before crystallization and its continued interrogation has been key to delineating the mechanistic bases for channel function (4–6). Although described 20 y ago (7), KcsA remains an orphan target so that studies with peptide toxins have required production of mutant channels with multiple mutations in the pore domain (8) or chimeras such as Kv1.3-KcsA, where the entire KcsA pore domain is replaced by the one in Kv1.3 (9).To isolate toxins for KcsA, a peptide library was designed with ∼1,562,750 variants via combinatorial permutation of sequences related to the sea anemone type I (SAK1) toxin ShK. Phage sorting was performed on purified, wild-type KcsA channels. Peptides expressed on the enriched phage were synthesized and studied by surface plasmon resonance (SPR) to characterize their binding to purified KcsA and by voltage-clamp electrophysiology to assess channel blockade. Hui1, a novel and specific inhibitor of KcsA, HmK, a natural and promiscuous blocker, and Hui1 mutants were evaluated to identify toxin segments and residues responsible for specificity and affinity and to discern the mechanism of channel inhibition. The 3D structure of Hui1 determined by NMR, the 1:1 stoichiometry of KcsA inhibition via a pore-directed mechanism, and the role of two, canonical “dyad” residues (Lys₂₁ and Tyr₂₂) in high-affinity binding all met expectations for a SAK1-type toxin. In contrast, the influence of permeant trans ions (those traversing the channel after entering from the opposite side of the membrane) on dissociation of Hui1 from its external binding site indicated that Arg₂₃, a residue with a side chain too bulky to fit snugly into the potassium conduction pore (10), was responsible for the voltage dependence of block rather than Lys₂₁. This unexpected role for Hui1-Arg₂₃ could reflect a new SAK1 binding orientation for the novel toxin; however, some models have located the ShK dyad Lys in the outer pore vestibule of Kv1.3 (11) rather than in the narrow portion of the conduction pathway (12). We posit that Hui1 binds and blocks like some, and perhaps most, natural SAK1 toxins.     

Bruxism-induced parotitis: A retrospective case series analysis

ABSTRACT

Objective: A group of patients with recurrent parotitis had clinical symptoms suggesting an obstruction of the glandular duct system, though without any signs of sialoliths or other ductal pathologies.

Methods: Clinical and radiological data of five patients with recurrent obstructive parotitis and long-standing definite bruxism were reviewed retrospectively. Ultrasonography, MRI, and sialendoscopy were performed for evaluation of ductal pathologies as the cause of glandular obstruction. Electromyography was used to evaluate the activity of masticatory muscles in all patients. The parotitis was treated with injection of botulinum toxin into the buccinator muscle.

Results: Dilatation of the Stensen’s duct proximal to the area of the buccinator muscle was shown in radiological examination with no evidence of ductal strictures. Electromyography revealed an abnormal activity of the ipsilateral buccinator muscle. All patients were free of disease after treatment of the buccinator muscle.

Discussion: Bruxism should be considered as a possible cause of recurrent parotitis.     

Induction of immune responses and prevention of alveolar bone loss by intranasal administration of mice with Porphyromonas gingivalis fimbriae and recombinant cholera toxin B subunit

INTRODUCTION: Adult periodontitis is initiated by specific periodontal pathogens represented by Porphyromonas gingivalis; however, an effective measure for preventing the disease has not yet been established. In this study, the effectiveness of a vaccine composed of fimbriae of P. gingivalis and recombinant cholera toxin B subunit (rCTB) was evaluated using BALB/c mice. METHODS: Fimbriae and rCTB were co-administered intranasally to BALB/c mice on days 0, 14, 21, and 28. On day 35, mice were sacrificed to determine immunoglobulin levels in serum, saliva, and nasal and lung extracts by enzyme-linked immunosorbent assay. The prevention effect of the vaccine on P. gingivalis-induced periodontitis in mice was evaluated by measuring alveolar bone loss. RESULTS: The rCTB significantly increased serum immunoglobulin (Ig)A levels when mice were administered with a minimal amount (0.5 microg) of the fimbrial antigen. The adjuvant effect on serum IgG production was indistinct because the minimal amount of the antigen still induced a large amount of IgG. In contrast to systemic responses, a fimbria-specific secretory IgA response was strongly induced by co-administration of rCTB and 0.5 microg fimbriae; the same amount of the antigen alone scarcely induced a response. Histopathological examination revealed IgA-positive plasma cells in the nasal mucosal tissue but no observable mast cells in the area. In addition, nasal administration of the fimbrial vaccine significantly protected the mice from P. gingivalis-mediated alveolar bone loss. CONCLUSION: Nasal vaccination with a combination of fimbriae and rCTB can be an effective means of preventing P. gingivalis-mediated periodontitis.     

消癌解毒方联合R-CHOP样方案治疗弥漫大B细胞淋巴瘤临床观察

目的 观察消癌解毒方联合R-CHOP样方案治疗初诊弥漫大B细胞淋巴瘤的临床疗效。方法 将40例弥漫大B细胞淋巴瘤患者采用随机数字表法随机分为治疗组与对照组,每组20例,治疗组采用消癌解毒方联合一线R-CHOP样方案,对照组单纯采用R-CHOP样方案,两组均化学治疗6个周期。比较两组患者临床疗效、生活质量变化及中医临床证候改善情况。结果 两组实体瘤疗效比较,差异无统计学意义（P＞0.05）。与治疗前比较,两组患者治疗后卡氏功能状态（Karnofsky performance status, KPS）评分显著升高(P＜0.05),中医证候积分显著降低（P＜0.05）,治疗组患者KPS评分升高程度和中医证候积分降低程度均显著大于对照组(P＜0.05)。两组患者不良反应发生率比较,差异无统计学意义(P＞0.05)。结论 消癌解毒方联合R-CHOP样方案治疗弥漫大B细胞淋巴瘤有助于改善患者临床症状,提高患者生活质量。     

泄浊解毒方对溃疡性结肠炎大鼠血清IL-1β、IL-10及结肠黏膜CD14的影响

目的:通过动物实验证实泄浊解毒方对溃疡性结肠炎(UC)大鼠治疗有效,并探讨其可能作用机制。方法:将40只实验大鼠随机分为空白对照组、模型对照组、柳氮磺吡啶组、中药高、低剂量组5组,除空白组外,其余4组均采用TNBS/乙醇联合造模法制造UC模型。分组灌胃治疗14 d后,分别检测大鼠血清IL-1β、IL-10、结肠黏膜CD14含量。结果:模型组较空白组IL-1β、CD14表达明显升高,IL-10表达明显下降,差异有显著性(P0.05);中药高、低剂量组、柳氮磺吡啶组较模型组IL-1β、CD14表达明显下降,IL-10表达明显升高,差异有显著性(P0.05);中药高剂量组较柳氮磺吡啶组、中药低剂量组IL-1β、CD14表达明显下降,IL-10表达明显升高,差异有显著性(P0.05)。结论:泄浊解毒方干预UC大鼠可降低血清IL-1β、升高血清IL-10、下调结肠黏膜CD14表达,这可能是其作用机制之一。     

论阿尔兹海默病“毒损脑络”病机的含义

王永炎院士提出"毒损脑络"理论,认为"毒损脑络"是阿尔兹海默病重要病机。"毒损脑络"理论中的"脑络",相当于现代医学的"血脑屏障及其载体功能";而Aβ属于中医的"毒"。阿尔茨海默病中西医病机可表述为:Aβ聚集(毒)可损害血脑屏障及其功能(脑络),而血脑屏障功能破坏(脑络)也会促进Aβ(毒)的脑内聚集。在临床应用"毒损脑络"治疗阿尔茨海默病过程中,除解毒、化瘀、化痰之外,补肾益气保护脑络功能也为常用治疗方法。     

Evaluation of Intradermal Injection of Botulinum Toxin A for Facial Lifting

BACKGROUND: Intradermal botolinum toxin A (BTXA) is an advanced technique that emerged in response to the increased demand for noninvasive facial lifting and skin rejuvenation. OBJECTIVE: We sought to evaluate the safety and efficacy of intradermal injections of BTXA for facial lifting. METHODS: Twenty-five female patients with mild symmetrical facial skin laxity were enrolled in this study. All patients were treated with BTXA in one side of the face while the other side was injected with normal saline. The response to treatment was assessed by two dermatologists who evaluated global photographs using a quartile grading scale (QGS). The patient self-assessment and satisfaction questionnaires were recorded. RESULTS: A highly significant difference was found between the side injected with BTXA and the saline injected side (control) (p<0.001). Facial lifting was achieved in 58.66 percent of the sides injected with BTXA. Forty-four percent of patients were very satisfied. Older patients showed better improvement than younger patients (p=<0.001). The results persisted for 16 weeks. No adverse effects were observed. CONCLUSION: Our results suggest that intradermal injection of BTXA could be a safe and effective therapeutic option for face lifting.