Many viruses, which infect algae, code for small membrane proteins with the characteristics of potassium channels. The peculiarity of these channels is that they have a monomer size of less than 100 amino acids. The structural simplicity and functional robustness of these miniature channels makes viral K+ channels very good model systems for understanding the basic molecular architecture of potassium channels. To learn more about structure/function correlates in these simple channel proteins I tried to generate a library of channel sequences. For this purpose, I searched for channel orthologues in environmental water samples and DNA databases.
Here I present some interesting new viral potassium channels from salt and fresh water viruses. Structural prediction algorithms indicate that the new channels from salt water viruses have the canonical α-helix folds, which are typical for the pore module of all known K+ channels. However, structural prediction algorithms failed to identify the expected transmembrane domains flanking the potassium channel pores. The fact that electrophysiological measurements confirmed an activity of these channels suggests that the transmembrane organization of these proteins is achieved in a different manner than in other K+ channels.
The newly identified viral K+ channels have unique functional properties: Two genes encode proteins, Kmpv12T and Kmpv1, of only 78 or 79 amino acid per unit, respectively. These are the smallest known K+ channels and this small size is presumably close to the absolute minimal size for a K+ channel. Both could be identified as functional channels by a combination of heterologous expression and electrophysiological measurements. In addition to these extra small channels from Micromonas sp. viruses also the function of Kmpvsp1 was confirmed. This channel exhibits unlike all other viral channels a pronounced inward rectification and some permeability to Na+. Kbpv1 from a Bathycoccus sp. virus and KotvRT from a Ostreococcus sp. virus could be identified as functional and selective potassium channels. KotvRT exhibits a steep voltage dependend Ba2+ and Cs+ block, which is similar to the Cs+ block of KcvNTS.
Two new channels were also isolated from fresh water Chlorella viruses. One of them, KcvGLND is from an evolutionarily interesting hybrid virus, which contains genes of SAG viruses and Pbi viruses. KcvNH is a channel from the KcvATCV-1 family, with interesting structure-function relations.
Some proteins like Kmpv12T have a channel-like structure but fail to generate a conductance after expression in HEK293 cells. After investigating the cellular distribution of GFP-tagged proteins we found that all the channels, which were positively identified in HEK293 cells in patch clamp recordings, were sorted into the secretory pathway and presumably from there to the plasma membrane. The channels which failed to generate currents in the plasma membrane of HEK293 cells were on the other hand predominantly sorted into the mitochondria or remained unsorted in the cytosol. When Kmpv12T was synthesized as representative for the latter channels in vitro and reconstituted into planar lipid bilayers it also generated typical potassium channel activity. Collectively, the data show that the majority of newly identified viral K+ channels generated, in spite of non-canonical structural features such as a small size and an unusual transmembrane domain architecture, potassium channel function. Some of the new channels exhibit an unusual sorting to the mitochondria. The finding that some of these proteins generate channel activity in planar lipid bilayers suggests that also these proteins are functional K+ channels.
| Identifer | oai:union.ndltd.org:tu-darmstadt.de/oai:tuprints.ulb.tu-darmstadt.de:7165 |
| Date | January 2018 |
| Creators | Siotto, Fenja |
| Source Sets | Technischen Universität Darmstadt |
| Language | English |
| Detected Language | English |
| Type | Ph.D. Thesis, NonPeerReviewed, info:eu-repo/semantics/doctoralThesis |
| Format | text |
| Rights | only the rights of use according to UrhG, info:eu-repo/semantics/openAccess |
| Relation | http://tuprints.ulb.tu-darmstadt.de/7165/ |
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