Structure and function of Paramecium bursaria chlorella virus-1 encoded K+ channel Kcv
Zusammenfassung der Projektergebnisse
The goal of the project was to extract fundamental structure/ function correlates from the miniature size, virus coded K+ channel Kcv. This was addressed by a combination of genetic, biochemical and computational analysis. A major result of the study was that all Chlorella viruses from the family of Phycodnaviridae coded for K+ channels. A new virus species even coded for a K+ channel and a water channel. The derived amino acid sequences from one virus species differed in some virus isolates in 5 (minimum) to 12 (maximum) positions from the reference channel. Functional studies of the relevant channel orthologs showed that some of the amino acid exchanges are responsible for drastic alterations in the function of the channel. In this context we found an amino acid position in the outer trans-membrane domain (TM1) which drastically affects the voltage sensitivity of the channel and which determined the sensitivity of the channel to Cs+ block. Such an important functional role of the TM1 domain was until then not known. The apparent significant function of TM1 was further examined by mutational studies in which the hydrophobic length of this domain was altered. Also interaction of TM1 with an upstream helical domain was examined. The functional analysis of the mutants and a complementary study of their structures by molecular dynamic simulations revealed an important interplay between the inner and outer transmembrane domains. Computational analysis and electrophysiological data are consistent with a model in which the entrance or exit of K+ from the cavity of the protein is determined by a stochastic formation and release of salt bridges between the two domains. The results of these experiments shade a new light on the functional role of the hydrophobic gate at the cytoplasmic side a K+ channel proteins; it occurs to be relevant also for other K+ channels. The function of the Kcv channel was further examined by purifying and functionally reconstituting the protein in planar lipid bilayers. The data reveal that the protein has an extraordinary stability as a tetramer; oligomerization of the isolated protein is in a very interesting way depending on cations. Only those cations, which permeate the channel are also able to protect the disintegration of the tetramer. The results of these experiments foster the hypothesis that an intimate relation between cations and channel protein in the selectivity filter determines the function of the channel. Some functional properties of the channel in the lilayer such as hyperactivity in Rb+ and transmembrane inhibition of K+ currents by Cs+ or Na* might be
Projektbezogene Publikationen (Auswahl)
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Gazzarrini, S., Kang, M., Epimashko, S., Van Etten, J.L. Dainty, J., Thiel, G., Moroni, A. (2006). Chlorella virus MT325 encodes water and potassium channels that interact synergistically. Proc. Natl. Acad. Sei. U.S.A. 103: 5355- 5360.
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Gazzarrini, S., Kang1 M., Van Etten, J.L., Tayefeh, S., Käst, S.M., DiFrancesco, D., Thiel, G., Moroni, A. (2004). Long-distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins. J. Biol. Chem. 279:28443-28449.
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Hertel, B., Horvath, F., Wodaia, B., Hurst, A., Moroni, A., Thiel, G. (2005). KAT1 inactivates at sub-millimolar concentrations of external potassium. J. Exp. Bot. 422:3103-3110.
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Hertel, B., Tayefeh, S., Mehmel, M., Käst, S.M., Van Etten, J., Moroni, A., Thiel, G. (2006). Elongation of outer transmembrane domain alters function of miniature K+channel Kcv. J. Membr. Biol. 210:21-29,
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Kang, M., Graves, M., Mehmel, M., Moroni, A., Gazzarrini, S., Thiel, G,, Gurnon, J., Van Etten, J.L. (2004). Genetic Diversity in Chlorella viruses flanking kcv, a gene that encodes a potassium ion channel protein. Virology 326:150-159.
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Kang, M., Moroni, A., Gazzarrini, S., DiFrancesco D., Thiel, G., Severino' M., Van Etten, J.L. (2004). Small potassium ion channel protein encoded by Chlorella viruses. Proc. Natl. Acad. Sei. U.S.A. 101:5318-5324.
