Chapter 3 : Deciphering KcsA as a K Channel Model

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As a member of the family Streptomycetaceae, is a gram-positive soil bacterium with a complex growth cycle. In the presence of nutrients this growth is initiated by the germination of spores. Giant liposome-protoplast vesicles derived from the mutant strain carrying the plasmid pKCS1 (with the K channel of streptomycetes A (KcsA) gene) showed ion channel activity under neutral pH (7.2) and asymmetric conditions. The negatively charged lipid phosphatidylglycerol (PG) and the nonbilayer lipid PE support tetramerization and membrane association of KcsA better than the zwitterionic bilayer lipid phosphatidylcholine (PC). In vitro studies using a transcription-translation system revealed that no tetramer is formed in a membrane-free reaction but only after the addition of inner membrane vesicles. With a coupled in vitro transcription-translation system, highest tetramerization was recorded in the presence of pure lipid vesicles, demonstrating that a phospholipid bilayer is the minimal requirement to form the KcsA tetramer. Polyhydroxybutyrate (PHB) and inorganic phosphate (polyP) are widely distributed among prokaryotic and eukaryotic organisms. The results of experimental and comparative structural data support the conclusion that the crystal structure of the tetrameric KcsA does not present the open state. The majority of the virus progeny is released about 8 h after infection. The current model assumes that Kcv is located in the internal membrane of the virus and will be inserted into the plasma membrane of the host cell. The hydration state of K ions and their permeation need to be reinvestigated.

Citation: Schrempf H. 2005. Deciphering KcsA as a K Channel Model, p 41-68. In Kubalski A, Martinac B (ed), Bacterial Ion Channels and Their Eukaryotic Homologs. ASM Press, Washington, DC. doi: 10.1128/9781555816452.ch3
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Figure 1

Features of and the generation of giant protoplasts. (A) Vegetative network of hyphae. (B) Protoplasts of were obtained by digesting (with lysozyme) the cell wall of the hyphae. (C) General scheme to fuse protoplasts with liposomes to generate giant protoplast vesicles. (D) Current recording at pH 7.2 under asymmetric conditions and the deduced histogram to present the proportion of closed and open channels (top). Current recordings and all point histograms after the addition of CsCl (bottom). (E) Current recordings (excised patch) under asymmetric conditions within ranging potentials.

Citation: Schrempf H. 2005. Deciphering KcsA as a K Channel Model, p 41-68. In Kubalski A, Martinac B (ed), Bacterial Ion Channels and Their Eukaryotic Homologs. ASM Press, Washington, DC. doi: 10.1128/9781555816452.ch3
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Figure 2

Characteristics of the deduced and the isolated KcsA protein as well as electrophysiological analysis of using a bilayer. (A) The characteristic regions of the KcsA protein deduced from the corresponding gene. (B) The KcsA protein assembles to a stable tetramer (left), which upon increase of temperature, can be resolved via intermediates (middle; above 70°C) to the monomeric form (right; melting point close to 85°C). (C) Generation of proteoliposomes and subsequent fusion with a bilayer. (D) Current recordings of a bilayer containing KcsA at pH 7.2 under asymmetric conditions. (E) Current recordings at different combinations of pH at and sites under symmetric conditions.

Citation: Schrempf H. 2005. Deciphering KcsA as a K Channel Model, p 41-68. In Kubalski A, Martinac B (ed), Bacterial Ion Channels and Their Eukaryotic Homologs. ASM Press, Washington, DC. doi: 10.1128/9781555816452.ch3
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Figure 3

Comparative electrophysiological characteristics of KcsA wild-type (WT) and mutant proteins generated after exchange of residues L81 and Y82. (A) The relative positioning of the relevant amino acids within KcsA. (B) Relative open probabilities of the channels (white bars, −100 mV; black bars, +100 mV). (C) Comparative sensitivity to TEA of WT and mutant channels (white bars, −100 mV; black bars, +100 mV). (D) Voltage dependence of WT and mutant channels of external TEA block. (E) Rectification properties of different proteins.

Citation: Schrempf H. 2005. Deciphering KcsA as a K Channel Model, p 41-68. In Kubalski A, Martinac B (ed), Bacterial Ion Channels and Their Eukaryotic Homologs. ASM Press, Washington, DC. doi: 10.1128/9781555816452.ch3
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