Báo cáo khoa học: Novel a-conotoxins from Conus spurius and the a-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

Novel α-conotoxins from Conus spurius and the α-conotoxin El share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors

Tác giả: Estuardo López-Vera, Manuel B. Aguilar, Emanuele Schiavon, Chiara Marinzi, Ernesto Ortiz, Rita Restano Cassulini, Cesar V. F. Batista, Lourival D. Possani, Edgar P. Heimer de la Cotera, Francesco Peri, Baltazar Becerril, Enzo Wanke

Lĩnh vực: Neurofarmacologia Marina, Neurobiologia Celular e Molecular, Biotecnologie e Bioscienze, Medicina Molecular e Bioprocesos.

Nội dung tài liệu: Nghiên cứu này tập trung vào việc phân lập, xác định cấu trúc và đánh giá hoạt tính của hai độc tố α-conotoxin mới, SrIA và SrIB, từ ốc biển Conus spurius. Các độc tố này là peptide 18 axit amin với trình tự mới. Nghiên cứu so sánh hoạt tính của chúng với α-conotoxin EI, một độc tố đối chứng, trên các thụ thể acetylcholine nicotinic (nAChRs) khác nhau (α1β1γδ, α4β2 và α3β4) bằng kỹ thuật patch-clamp. Kết quả cho thấy SrIA, SrIB và một chất tương tự tổng hợp của chúng có ái lực cao trong việc điều hòa và tăng cường phản ứng cholinergic của thụ thể α1β1γδ và α4β2, đồng thời thể hiện ái lực thấp trong việc ức chế các thụ thể này. Ngược lại, EI cho thấy ái lực cao trong việc ức chế các thụ thể α1β1γδ, α3β4, và α4β2. Các đặc tính độc đáo này làm cho các độc tố này trở thành công cụ hữu ích để nghiên cứu tương tác độc tố-thụ thể và phát triển thuốc điều trị.

Mục lục chi tiết:

• Keywords: α-conotoxin; conotoxins; Conus spurius; nicotinic receptor; potentiation
• Correspondence
• Present address
• Abstract
• a-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [γ15E]SrIB) were compared to those exerted by the α4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing α1β1γδ, α4β2 and α3β4 nicotinic acetylcholine receptors. At high concentrations (10 μm), the peptides SrIA, SrIB and [γ15E]SrIB showed weak blocking effects only on α4β2 and α1β1γδ subtypes, but EI also strongly blocked α3β4 receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of α1β1γδ and α4β2 nicotinic acetylcholine receptors if briefly (2–15 s) applied at concentrations several orders of magnitude lower (EC50, 1.78 and 0.37 nm, respectively). These results suggest not only that the novel α-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both α1β1γδ and α4β2 nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.
• Conotoxins are small, disulfide-rich peptides that have been isolated from Conus, a large genus of predatory marine snails. The primary structures of more than 100 conotoxins have been determined and classified into gene superfamilies on the basis of the amino acid sequences of the signal peptides of their precursors.
• Abbreviations
• Structure-function relationship for SrIA, SrIB, and El
• The physiologic role of the SrI and El α-conotoxins
• Experimental procedures
• Specimen collection and venom extraction
• Peptide purification by RP-HPLC
• Amino acid sequence
• MS analysis
• Determination of disulfide bridges
• cDNA cloning
• Synthesis of peptides [γ15E]SrIB and El
• Solid-phase peptide synthesis
• Formation of the first disulfide bond
• Acm group removal and formation of the second disulfide bond
• Electrophysiology
• Cell culture
• Solutions
• Patch-clamp recordings
• Acknowledgements
• References