Abstract
Viroporins are small virus-encoded ion channel proteins. Most viroporins are monovalent selective cation channels, with few showing the ability to conduct divalent cations, like calcium (Ca2+). Nevertheless, some viroporins are known to disrupt host cell Ca2+ homeostasis, which is critical for virus replication and pathogenesis. Rotavirus nonstructural protein 4 (NSP4) is an endoplasmic reticulum transmembrane glycoprotein that has a viroporin domain (VPD), and NSP4 viroporin activity elevates cytosolic Ca2+ in mammalian cells. The goal of this study was to demonstrate that the NSP4 VPD forms an ion channel and determine whether the channel can conduct Ca2+. Using planar lipid bilayer and liposome patch clamp electrophysiology, we show that a synthetic peptide of the NSP4 VPD has ion channel activity. The NSP4 VPD was selective for cations over anions and channel activity was observed to have both well-defined "square top" openings as well as fast current fluctuations, similar to other viroporins. Importantly, the NSP4 VPD showed similar conductance of divalent cations (Ca2+ and Ba2+) as monovalent cations (K+), but a viroporin defective mutant lacked Ca2+ conductivity. These data demonstrate that the NSP4 VPD is a Ca2+-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca2+ homeostasis.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Barium / chemistry
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Barium / metabolism*
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Calcium / chemistry
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Calcium / metabolism*
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Calcium Channels / chemistry
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Calcium Channels / genetics
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Calcium Channels / metabolism*
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Cholesterol / chemistry
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Cholesterol / metabolism
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Glycoproteins / chemistry
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Glycoproteins / genetics
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Glycoproteins / metabolism*
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Ion Transport
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Membrane Potentials / physiology
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Mutation
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Patch-Clamp Techniques
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Peptides / chemistry
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Peptides / genetics
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Peptides / metabolism*
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Phosphatidylcholines / chemistry
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Phosphatidylcholines / metabolism
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Proteolipids / chemistry
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Proteolipids / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Rotavirus / chemistry
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Rotavirus / metabolism
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Structure-Activity Relationship
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Toxins, Biological / chemistry
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Toxins, Biological / genetics
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Toxins, Biological / metabolism*
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Unilamellar Liposomes / chemistry
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Unilamellar Liposomes / metabolism
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Viral Nonstructural Proteins / chemistry
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Viral Nonstructural Proteins / genetics
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Viral Nonstructural Proteins / metabolism*
Substances
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Calcium Channels
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Glycoproteins
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NS28 protein, rotavirus
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Peptides
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Phosphatidylcholines
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Proteolipids
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Recombinant Proteins
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Toxins, Biological
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Unilamellar Liposomes
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Viral Nonstructural Proteins
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proteoliposomes
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Barium
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1,2-diphytanoylphosphatidylcholine
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asolectin
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Cholesterol
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Calcium