title: Structure and function analysis of two channel forming membrane proteins of medical importance – The lysosomal Transmembrane Protein 175 and the Envelope Protein of SARS-CoV-2
Creators: name: Schulze, Tobias
Publisher: name: UNSPECIFIED
Year: 2022

description: In this work, two channel proteins of medical importance were electrophysiologically investigated and characterized. In the first part of this work, the lysosomal TMEM175 channel was in the focus of the studies. TMEM175 is associated with the development of Parkinson’s disease (PD) through a yet unknown mechanism, underscoring the importance of this channel to regulation of cellular homeostasis. The family of TMEM175 proteins in general constitutes a recently discovered type of K+ channels that are important for autophagosome turnover and lysosomal pH regulation. From a structural point of view, TMEM175 channels lack the typical P-loop type selectivity filter, a hallmark of all canonical K+ channels. This raises the question on how K+ selectivity is achieved in TMEM175 channels. In this study, new insights gained by the X-ray structure of a closed bacterial TMEM175 channel are used in combination with electrophysiological methods to address this very question. By performing mutational studies and subsequent electrophysiological investigations, Thr38 of MtTMEM175 as well as the corresponding layer of threonines in the human homolog (hTMEM175) were found to play a pivotal role in K+ selectivity. The data suggests this mechanism to be a general explanation for K+ selectivity in TMEM175 channels. An additional layer in the hTMEM175 comprising two serines further increases selectivity and renders this channel sensitive to blockers like 4-aminopyridine and Zn2+. The combination of structural data, mutations and electrophysiological measurements indicate that large hydrophobic side chains occlude the pore, forming a physical gate in TMEM175 channels. Channel opening of MtTMEM175 by an iris-like motion simultaneously relocates the gate and exposes the otherwise concealed selectivity filter to the pore lumen. Close scrutiny of closed- and open-state hTMEM175 cryo-EM structures furthermore provides a coherent hypothesis on a gating mechanism based on charged residues at the extracellular pore entrance. In the second part of this work, the envelope protein of SARS-CoV-2 and its effects on the homeostasis of the host cell of the virus were studied. SARS-CoV-2 produces in host cells large amounts of envelope proteins (Ep-CoV-2). To mimic its pathophysiological impact, Ep-CoV-2 was expressed in mammalian cells and the effects on signaling parameters monitored. We detected fluorescent tagged Ep-CoV-2 in the endoplasmic reticulum and trace amounts in the plasma membrane. Wild-type (wt) Ep-CoV-2 and, to a lesser extent, its mutants (N15A, V25F) and the isolated transmembrane domains corrupted major signaling cascades in cells causing elevated intracellular Ca2+ as well as pH and membrane depolarization. These Ep-CoV-2-triggered effects, which potentially contribute to the pathogenesis of the viral protein, seem to result from an ion-channel activity. Two independent assays, functional reconstitution of Ep-CoV-2 in artificial membranes and rescue of K+-deficient yeast mutants, confirm that Ep-CoV-2 generates a cation-conducting channel with a low unitary conductance and a complex ion selectivity. All results together suggest that inhibitors of this channel function can provide cell protection and virostatic effects.