Reconstitution and functional characterisation of simple channel proteins in the planar lipid bilayer.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2010)
Dissertation Michael Henkel / Fachbereich Biologie -
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|Item Type:||Ph.D. Thesis|
|Title:||Reconstitution and functional characterisation of simple channel proteins in the planar lipid bilayer|
The present study describes the electrophysiological characterisation of different primitive channel-forming proteins, respectively peptides. Using the so-called `planar lipid bilayer technique`, which is a maximally reduced system for the functional reconstitution and electrophysiological characterization of purified channel proteins, protein-/ peptide-mediated single channel currents were measured. Depending on defined ion concentrations in the bath solution on cis- and trans-side of a membrane, typical properties of the reconstituted channels such as current/ voltage relationships, open probability and selectivities could be determined. Chapter 2 deals with the wildtype and two different mutants of Kcv, a tetrameric K+ channel, which is encoded by the Paramecium bursaria Chlorella Virus-1 (PBCV-1). The data reveal that the subtle mutation of one amino acid (T->S of residue 63), which lies in the selectivity filter next to the cavity, almost completely reverses the ability of the wildtype to be blocked by Ba2+. Furthermore, the mutation causes a considerable increased open probability, whereas the channel rarely reaches the maximal conductance level; mostly the channel opens to different subconductances. These subconductances probably reflect different kinetic states of the channel; simulations based on Markov models reveal that a very fast gating in combination with a limited registration of the channel gating can be responsible for apparent subconductances. The altered function of the mutant must be due a sensitive change in the protein structure because a mutation of a second, adjacent amino acid is able to recover the properties of the wildtype. Chapter 3 deals with different versions of the PB1-F2 protein which are encoded by different Influenza A viruses. It was already described in literature that this protein is able to augment the conductance in the planar lipid bilayer. The absence of discrete conductance fluctuations suggested that PB1-F2 is not a canonical channel. However, the instant study shows that synthetic peptide analogues of PB1-F2 generate canonical channel function in the planar lipid bilayer. In combination with fluorometric studies, the electrical data reveal that the PB1-F2-generated channels possess two discrete conductance levels and unspecifically conduct cations and anions. Chapter 4 deals with phospholamban, a protein whose function as modulator of the sarco-/ endoplasmatic Ca-ATPase (SERCA) was described already previously. For a long time it was known that the monomer of phospholamban is in equilibrium with the pentameric form, whereas the latter one is considerably more stable. It is a matter of discussion whether the pentamer has a channel function. Impedance measurements in so-called `supported nano-BLMs`, in which the protein was reconstituted and which were performed in cooperation with the group of Moncelli at the institute for chemistry of the University of Florence, show that phospholamban indeed induces typical ion channel fluctuations in membranes. The reconstitution of phospholamban in the planar lipid bilayer supports the hypothesis of a phospholamban-mediated channel function with two discrete cation-selective conductance levels at 16 pS and 27 pS.
|Place of Publication:||Darmstadt|
|Uncontrolled Keywords:||single ion channel reconstitution, planar lipid bilayer, electrophysiology, PB1-F2, Kcv, Phospholamban|
|Classification DDC:||500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin, Gesundheit
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
|Divisions:||10 Department of Biology|
|Date Deposited:||18 Sep 2010 10:28|
|Last Modified:||07 Dec 2012 11:58|
|Referees:||Thiel, Prof. Dr. Gerhard and Galuske, Prof. Dr. Ralf|
|Refereed:||3 September 2010|