{"title":"Structural Dynamics of the Slide Helix of Inactive/Closed Conformation of KirBac1.1 in Micelles and Membranes: A Fluorescence Approach.","authors":"Arpan Bysack, Chandrima Jash, H Raghuraman","doi":"10.1007/s00232-024-00335-y","DOIUrl":null,"url":null,"abstract":"<p><p>Inward rectifying potassium (Kir) channels play a critical role in maintaining the resting membrane potential and cellular homeostasis. The high-resolution crystal structure of homotetrameric KirBac1.1 in detergent micelles provides a snapshot of the closed state. Similar to micelles, KirBac1.1 is reported to be in the inactive/closed conformation in POPC membranes. The slide helix of KirBac1.1 is an important structural motif that regulates channel gating. Despite the importance of slide helix in lipid-dependent gating, conflicting models have emerged for the location of slide helix and its structural dynamics in membrane mimetics is poorly understood. Here, we monitored the structural dynamics of the slide helix (residues 46-57) of KirBac1.1 in both DM micelles and POPC membranes utilizing various site-directed fluorescence approaches. We show, using ACMA-based liposome-flux assay, the cysteine mutants of the slide helix are not functional, ensuring the inactive/closed conformation in POPC membranes similar to wild-type channel. Time-resolved fluorescence and water accessibility measurements of NBD-labeled single-cysteine mutants of slide-helix residues suggest that the location of the slide helix at the interfacial region might be shallower in membranes compared to micelles. Interestingly, the slide helix of KirBac1.1 is more dynamic in the physiologically relevant membrane environment, which is accompanied by a differential hydration dynamics throughout the slide helix. Further, REES and lifetime distribution analyses suggest significant changes in conformational heterogeneity of the slide helix in membrane mimetics. Overall, our results give an insight into how membrane mimetics affect the organization and dynamics of slide helix of the closed state of KirBac1.1, and highlight the importance of lipid-protein interactions in membranes.</p>","PeriodicalId":50129,"journal":{"name":"Journal of Membrane Biology","volume":" ","pages":""},"PeriodicalIF":2.3000,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Membrane Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s00232-024-00335-y","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Inward rectifying potassium (Kir) channels play a critical role in maintaining the resting membrane potential and cellular homeostasis. The high-resolution crystal structure of homotetrameric KirBac1.1 in detergent micelles provides a snapshot of the closed state. Similar to micelles, KirBac1.1 is reported to be in the inactive/closed conformation in POPC membranes. The slide helix of KirBac1.1 is an important structural motif that regulates channel gating. Despite the importance of slide helix in lipid-dependent gating, conflicting models have emerged for the location of slide helix and its structural dynamics in membrane mimetics is poorly understood. Here, we monitored the structural dynamics of the slide helix (residues 46-57) of KirBac1.1 in both DM micelles and POPC membranes utilizing various site-directed fluorescence approaches. We show, using ACMA-based liposome-flux assay, the cysteine mutants of the slide helix are not functional, ensuring the inactive/closed conformation in POPC membranes similar to wild-type channel. Time-resolved fluorescence and water accessibility measurements of NBD-labeled single-cysteine mutants of slide-helix residues suggest that the location of the slide helix at the interfacial region might be shallower in membranes compared to micelles. Interestingly, the slide helix of KirBac1.1 is more dynamic in the physiologically relevant membrane environment, which is accompanied by a differential hydration dynamics throughout the slide helix. Further, REES and lifetime distribution analyses suggest significant changes in conformational heterogeneity of the slide helix in membrane mimetics. Overall, our results give an insight into how membrane mimetics affect the organization and dynamics of slide helix of the closed state of KirBac1.1, and highlight the importance of lipid-protein interactions in membranes.
期刊介绍：
The Journal of Membrane Biology is dedicated to publishing high-quality science related to membrane biology, biochemistry and biophysics. In particular, we welcome work that uses modern experimental or computational methods including but not limited to those with microscopy, diffraction, NMR, computer simulations, or biochemistry aimed at membrane associated or membrane embedded proteins or model membrane systems. These methods might be applied to study topics like membrane protein structure and function, membrane mediated or controlled signaling mechanisms, cell-cell communication via gap junctions, the behavior of proteins and lipids based on monolayer or bilayer systems, or genetic and regulatory mechanisms controlling membrane function.
Research articles, short communications and reviews are all welcome. We also encourage authors to consider publishing ''negative'' results where experiments or simulations were well performed, but resulted in unusual or unexpected outcomes without obvious explanations.
While we welcome connections to clinical studies, submissions that are primarily clinical in nature or that fail to make connections to the basic science issues of membrane structure, chemistry and function, are not appropriate for the journal. In a similar way, studies that are primarily descriptive and narratives of assays in a clinical or population study are best published in other journals. If you are not certain, it is entirely appropriate to write to us to inquire if your study is a good fit for the journal.
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