{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T03:47:59Z","timestamp":1760240879503,"version":"build-2065373602"},"reference-count":34,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2019,10,16]],"date-time":"2019-10-16T00:00:00Z","timestamp":1571184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["81872794","31760249","81660585"],"award-info":[{"award-number":["81872794","31760249","81660585"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Major International Joint Research Project of National Natural Science Foundation of China","award":["81420108028"],"award-info":[{"award-number":["81420108028"]}]},{"name":"Hainan University Youth foundation","award":["hdkyx201725"],"award-info":[{"award-number":["hdkyx201725"]}]},{"name":"Changjiang Scholars and Innovative Research Team in University Grant","award":["IRT_15R15"],"award-info":[{"award-number":["IRT_15R15"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>\u03b1-Conotoxins (\u03b1-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an \u03b14\/7-CTx, mainly blocks \u03b11\u03b21\u03b4\u03b5 nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse \u03b11\u03b21\u03b4\u03b5 nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse \u03b11\u03b21\u03b4\u03b5 nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the \u03b11\u03b21\u03b4\u03b5 nAChR, and showed loss of activity on \u03b13\u03b22 and \u03b13\u03b24 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure\u2013activity relationship (SAR) of EI revealed that the \u201cArg1\u2013Asn2\u2013Hyp3\u201d residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue \u25b31\u20133 EI caused a total loss of activity at the \u03b11\u03b21\u03b4\u03b5 nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue \u25b31\u20133 EI for \u03b11\u03b21\u03b4\u03b5 nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse \u03b11\u03b21\u03b4\u03b5 nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.<\/jats:p>","DOI":"10.3390\/toxins11100603","type":"journal-article","created":{"date-parts":[[2019,10,17]],"date-time":"2019-10-17T04:46:06Z","timestamp":1571287566000},"page":"603","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Identification of Crucial Residues in \u03b1-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor"],"prefix":"10.3390","volume":"11","author":[{"given":"Jiong","family":"Ning","sequence":"first","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jie","family":"Ren","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yang","family":"Xiong","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yong","family":"Wu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Manqi","family":"Zhangsun","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dongting","family":"Zhangsun","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8830-4954","authenticated-orcid":false,"given":"Xiaopeng","family":"Zhu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9358-5970","authenticated-orcid":false,"given":"Sulan","family":"Luo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Tropical Biological Resources, Ministry of Education, Key Lab for Marine Drugs of Haikou, School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,10,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1124\/pr.111.005322","article-title":"Conus venom peptide pharmacology","volume":"64","author":"Lewis","year":"2012","journal-title":"Pharmacol. 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