The role of the SARS‐CoV‐2 envelope protein as a pH‐dependent cation channel

Abstract

A novel human coronavirus known as severe acute respiratory syndrome (SARS)-coronavirus (CoV) 2 was isolated in Wuhan (China), and there followed an epidemic outbreak that started in December 2019. Coronaviruses include four main structural proteins: nucleocapsid protein (N), spike protein (S), membrane protein (M) and envelope protein (E) (McClenaghan et al. 2020). SARS-CoV-2 E protein is a small integral membrane protein that has three main domains: the hydrophilic N-terminal domain, the hydrophobic transmembrane domain and the hydrophilic C-terminal region. Several studies have emphasized the importance of the hydrophobic transmembrane domain, which self-interacts to form a pentameric ion pore that has shown little or no ion selectivity. Thus it might act as a viroporin during virus infection and/or release (Sarkar & Saha, 2020). Topological analysis of SARS-CoV-2 E protein has shown that the N-terminus is localized within intracellular organelles, whereas the C-terminus is localized in the cytoplasm. The E protein of SARS-CoV-1 has properties consistent with ion channel activity, and importantly, it shares 95% sequence identity with SARS-CoV-2 E. Together with functional studies of SARS-CoV-2 E in bacteria and in bilayer recordings, this information inspired Cabrera-Garcia et al. (2021), in a recent study published in The Journal of Physiology, to propose that SARS-CoV-2 E protein forms a cation channel. In the study by Cabrera-Garcia et al. (2021), an innovative strategy was used to express SARS-CoV-2 E protein in mammalian NIH 3T3 and HEK 293S cells, which allowed the characterization of its ion channel activity. They synthesized and subcloned three constructs in a pcDNA3 vector: (i) a wild-type (WT) construct, which encoded E protein targeted to the intracellular site (i.e. SARS2-E-mKate2); (ii) a plasma membrane (PM) construct, which encoded E protein targeted to the PM (i.e. SARS2-E-Ala6PBM-PM-mKate2); and (iii) an untagged PM construct that lacked the fluorescent tag, mKate2 (i.e. SARS2-E-Ala6PBM-PM). The NIH 3T3 cells were transfected with the WT (SARS2-E-mKate2) construct, and then imaged with inclusion of the fluorescent pH-sensitive indicator DND-189, to determine whether intracellular expression of E protein affected the pH of the internal organelles. With increased E protein expression, they reported a time-dependent increase in the internal pH of intracellular organelles, including in particular lysosomes and the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). Furthermore, membrane currents were measured for HEK 293S cells expressing the WT and PM constructs using whole-cell patch clamp to investigate E protein pore-forming activity. As expected, larger currents were obtained for the cells transfected with the PM construct, which confirmed that the membrane currents were associated with PM expression of E protein. The expression of the PM construct was also confirmed by measurements of membrane capacitance. HEK293S cells transfected with the PM construct had greater mean membrane capacitance and inward and outward current amplitudes. The normalized current density that was contributed by E protein was approximately ±1 pA/pF. Electrophysiological measurements were also carried out with HEK 293S cells transfected with the PM construct to determine the pH-dependent activity of expression of E protein as an ion channel. Here, they looked at changes in extracellular pH,which confirmed the voltage dependence of the ion current through ion channels formed by E protein (i.e. the E current). The E current was approximately linear at pH 7.4, whereas at pH 6.0 it showed inward rectification. At pH 8.0 the E current was reduced over a range of holding potentials. Changes in the intracellular and extracellular solutions facilitated the study of the selectivity of the ion channels formed by E protein. Na+ and K+ passed through these ion channels, while Cl− showed little ion channel permeation, and the bulky N-methyl-d-glucamine+ cation did not permeate through the channel. These observations led Cabrera-Garcia et al. (2021) to conclude that E protein forms an ion channel that is permeable to small monovalent cations, including Na+ and K+. In addition, they performed two-electrode voltage clamp current recordings in Xenopus laevis oocytes injected with cRNA encoding WT or untagged PM E protein constructs. At pH 7.5, PM E protein produced linear currents over the entire voltage range examined (−100 to 70 mV). Importantly, the current amplitude increased with the amount of PM cRNA injected. Compared to PM E protein, WT E protein did not produce significant currents, as expected. After changing the pH of the bath solution, the mean peak current observed at pH 6.0 was greater than that observed at pH 9.0, whereas no significant currents were observed at pH 6.0 in non-injected oocytes. E protein has been implicated in various stages of the coronavirus life cycle, including virus assembly, budding, envelope formation and release. It interacts with other viral and cellular proteins, and has several molecular functions, including its putative role as an ion channel (McClenaghan et al. 2020; Singh Tomar & Arkin, 2020). Initially, studies provided structural insights into the putative mechanism of action of E protein as a conformation-dependent ion channel that is permeable toH+ ions. Now theseWT and PM constructs designed by Cabrera-Garcia et al. (2021) have enabled functional investigation of E protein ion channel activity, and its influence on intracellular proton homeostasis. Recent studies have shown that only a small proportion of E protein that is expressed during SARS-CoV-2 infection is incorporated into the virion envelope. The rest is localized in the ER–Golgi complex region, and in particular, in the