The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel

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.