SARS-CoV-2 E and 3a Proteins Are Inducers of Pannexin Currents

Abstract

Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses induced by these proteins. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly acquired round shapes that detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this by using flow cytometry. In adhering cells expressing E or 3a protein, the whole-cell currents were not different from those of the control, suggesting that E and 3a proteins are not plasma membrane viroporins. In contrast, recording the currents on detached cells uncovered outwardly rectifying currents much larger than those observed in the control. We illustrate for the first time that carbenoxolone and probenecid block these outwardly rectifying currents; thus, these currents are most probably conducted by pannexin channels that are activated by cell morphology changes and also potentially by cell death. The truncation of C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent these outwardly rectifying currents. This suggests distinct pathways for the induction of these cellular events by the two proteins. We conclude that SARS-CoV-2 E and 3a proteins are not viroporins expressed at the plasma membrane.

Electrophysiological Impact of SARS-CoV-2 Envelope Protein in U251 Human Glioblastoma Cells: Possible Implications in Gliomagenesis?

“…2024, 25, x FOR PEER REVIEW 5 of 17 endogenous voltage-dependent potassium current (Kv) reported in HEK293 [31]. These data, however, do not exclude the presence of a very small viroporin aspecific cationic current as reported by other groups [20,21].…”

“…Intracellular calcium studies by FURA-2 highlighted, in the U251 line, a trend of increased intracellular calcium in the U251@Env that could explain the increase in IKCa- and BKCa-activated calcium currents. The increase in intracellular calcium could be due to the following: (i) calcium conduction through viroporin-formed by protein E expression mainly in intracellular membranes (see [ 21 ] but also [ 20 ]); (ii) the activation of endogenous calcium permeating cation channels such as those of the TRP family, which have been seen to be activated by the WNT pathway [ 42 ]; and (iii) disruption in intracellular calcium clearance due to the depolarization of the inner mitochondrial potential [ 43 ]. Further studies are needed to understand what signals are involved in the disruption of calcium homeostasis and which calcium potassium currents are promoted upon by protein E.…”

“…There are several issues regarding the membrane incorporation of the E protein and the plasma membrane current associated with its expression in different kinds of model cells, and the activity of E protein as an ion channel is still not fully understood [ 19 ]. While some authors showed a cationic channel activity for E protein [ 20 ], recently, it was hypothesized that the recorded currents are connected to the induction of endogenous pannexin channels [ 21 ].…”