Rectification of voltage-gated plant K+ channels
Final Report Abstract
The two prototypes of plant voltage-gated K+ channels, KAT1 and SKOR from "Arabidopsis thaliana", share ~28% identity at the amino acid level but exhibit fundamentally different functional properties. Whereas KAT1 is opening with negative-going, hyperpolarizing voltages and rectifies the K+ flux into the cell (Kin channel), SKOR activates upon depolarization and allows K+ efflux, only (Kout channel). Based on KAT1 and SKOR, a synthetic Kin channel (SKIN) and a synthetic Kout channel (SKOUT) were generated that share an identity of 82.5% at the protein level and of 90% at the DNA level. The design of the synthetic SKIN and SKOUT clones allowed to generate a cDNA library with >30.000.000 different SKIN/SKOUT chimeras. The SKIN-SKOUT library was exploited by using a K+ uptake-deficient strain of the yeast Saccharomyces cerevisiae. The library was screened for functional complementation of the yeast growing on medium with low K+ concentrations. Among 43 million transformants, 79 individual SKIN-SKOUT chimeras could be identified that enable yeast growth on low K+ medium. After expression in "Xenopus" oocytes, the identified chimeras segregated into two types with respect to their electrophysiological features. They were either inward-rectifying as SKIN or they lost the rectification properties and were open in the entire testable voltage-interval. The library design enabled also to easily generate a desired chimera for the expression in "Xenopus" oocytes. Several of the different segments in SKIN and SKOUT were swapped and the resulting chimeras were directly investigated in electrophysiological studies. Four different categories of results were obtained: chimeras 1) were inward-rectifying, 2) were outward-rectifying, 3) lost their rectification properties, or 4) lost their functionality. In a subsequent approach, combinations of exchanges were tested. Here, in several cases the combination of exchanges that alone resulted in rectifying channels, generated either non-functional or non-rectifying chimeras. Interestingly, however, also the inverse could be achieved by the combinatorial approach. The combination of three domain exchanges resulted in a Kin channel while the single exchanges rendered the chimeras non-rectifying. These results indicate that the rectification property is hidden in a complex 3D-puzzle of the channels’ architecture. To overcome the limitation of missing structural information 3D-homology models of KAT1, SKOR, SKIN and SKOUT were generated on the basis of animal Kv channels.
Publications
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(2010) “Molecular modeling of extracellular pH sensor of the KAT1 potassium channel”. Regulation of Plant Growth (RoPG) Conference 2010, Potsdam, Germany
González W, Riedelsberger J, Morales-Navarro SE, González-Nilo FD, Dreyer I
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(2010) “Rectification of voltage-gated plant potassium channels”. 15th International Workshop on Plant Membrane Biology (IWPMB), Adelaide, Australia
Riedelsberger J, Dreyer I
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Potassium channels in plant cells. The FEBS Journal, Vol. 278. 2011, Issue 22, pp. 4293–4303.
Dreyer I., Uozumi N.
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(2012) “Structural approach to study the voltage gating of potassium channels in Arabidopsis thaliana”. Biología Vegetal, Pucón, Chile
Morales-Navarro SE, Riedelsberger J, Dreyer I, Gonzalez W
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(2012) “The structural puzzle of rectification of voltage-gated plant potassium channels”. Biología Vegetal, Pucón, Chile
Riedelsberger J, Gonzalez W, Riadi G, Dreyer I
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(2012) “The structural puzzle of rectification of voltage-gated plant potassium channels”. MPI-Chile 2012, Berlin, Germany
Riedelsberger J, Riadi G, Dreyer I, Gonzalez W
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The pH-sensor of the plant K+ uptake channel KAT1 is built of a sensory cloud rather than of single key amino acids. Biochemical Journal, Vol. 442.2012, Issue 1, pp. 57–63.
Gonzalez, W., Riedelsberger, J., Morales-Navarro, S.E., Caballero, J., Alzate-Morales, J.H., González-Nilo, F.D., and Dreyer, I.
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(2013) “The structural puzzle of rectification of voltage-gated plant potassium channels”. 16th International Workshop on Plant Molecular Biology (IWPMB), Kurashiki, Japan
Riedelsberger J, Gonzalez W, Riadi G, Dreyer I
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The role of K+ channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana. Frontiers in Plant Science, Vol. 4.2013, 224.
Sharma T, Dreyer I, Riedelsberger J
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Voltage-Sensor Transitions of the Inward-Rectifying K+ Channel KAT1 Indicate a Latching Mechanism Biased by Hydration within the Voltage Sensor. Plant Physiology, Vol. 166. 2014, Issue 2, pp. 960-975.
Lefoulon C., Karnik R., Honsbein A., Gutla P.V., Grefen C., Riedelsberger J., Poblete T., Dreyer I., Gonzalez W., Blatt M.R.
