Synthesis of Modified Biomolecules to Investigate Neuronal Processes
Patrick Menzel
Doctoral thesis
Date of Examination:
2020-12-22
Date of issue:
2021-04-30
Advisor:
Diederichsen, Ulf Prof. Dr.
Referee:
Diederichsen, Ulf Prof. Dr.
Referee:
Steinem, Claudia Prof. Dr.
Referee:
Jahn, Reinhard Prof. Dr.
Referee:
Rizzoli, Silvio Prof. Dr.
Referee:
Frauendorf, Holm Dr.
Referee:
John, Michael Dr.
Referee:
Glöggler, Stefan Dr.
Persistent Address: http://hdl.handle.net/21.11130/00-1735-0000-0008-5810-3
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Abstract
English
Action potentials are an essential process for signal transmission in excitable cells. At chemical synapses, the action potential triggers the release of neurotransmitters from synaptic vesicles to transmit the signal to the target cells. After signal transmission, the neurotransmitters are refilled into synaptic vesicles. The uptake mechanism of the primary excitatory neurotransmitter glutamate is mediated by the vesicular glutamate transporter (VGLUT). However, the efficiency of glutamate transport by VGLUT is tightly regulated by the extravesicular and luminal Cl- concentrations. Despite many years of research, quantitative measurements of the Cl- conductance through VGLUT and the regulation of neurotransmitter uptake are still under discussion. For unravelling of the complex dependence between Cl- and glutamate transport, fluorescent Cl- sensors were developed, which allow measurements of the intravesicular Cl- concentration. The sensor was labelled with a Cl- sensitive BAC fluorophore and the insensitive AlexaFluor 647 dye. This combination of dyes allows a ratiometric measurement of the Cl- concentrations, independent of the experimental parameters. The BAC fluorophore, thereby, has the advantage of Cl- sensitivity within the physiological range, pH insensitivity as well as prolonged excitation and emission wavelengths compared to Cl- sensitive protein reporters. The backbone of the sensor, which connects both fluorophores, is composed of a PNA-DNA scaffold (PNA = peptide nucleic acid), combining the sensing, normalising and stabilisation module. The PNA-DNA backbone was further modified with membrane anchors, for enhanced incorporation of the Cl- sensor into the luminal site of the liposome. Modification with a protease cleavage sequence or affinity tag allows the specific removal of extravesicular orientated sensors from the liposome. For VGLUT dependent measurements of Cl- concentration, liposomes were co reconstituted with a bacterial proton pump (TF0F1) and VGLUT. Therefore, incorporation of the Cl- sensor allows direct quantification and kinetic measurement of Cl- in- and efflux through VGLUT in a controllable system. Apart from investigations of Cl- dependence during the neurotransmitter uptake of glutamate through VGLUT, an attempt of specific blockage of voltage-gated sodium (NaV) channels was studied in chapter two of this thesis. NaV channels are key players of the initiation and propagation of action potentials. Dysfunctions of the nine different isoforms are linked to diseases of the nervous system, heart rhythm and skeletal muscles contraction. However, especially malfunctions of the NaV1.2 channel subtype are associated with neurological disorders. For further understanding of the disease development and progression, improved visualisation of the NaV1.2 channels are essential. Therefore, new fluorescently labelled inhibitors with high affinity and specificity are required. Screening studies of neurotoxins from venomous cone snails revealed the µ-conotoxin SIIIA, which blocks the NaV1.2 channel with nanomolar affinity and the skeletal NaV1.4 channel with submicromolar affinity. The synthesis and challenging folding of the µ-conotoxin SIIIA could be improved by using a regioselective folding strategy with orthogonal thiol-protecting groups. After the successful formation of the native µ-SIIIA, the molecule was labelled with an AlexaFluor 488 fluorophore enabling channel visualisation. For activity measurements with the µ-conotoxin SIIIA, a caging approach was developed, which precisely allows NaV channel inhibition upon photocleavage of the caging group.
Keywords: VGLUT; Cl- Sensor; Peptide nucleic acid; His-tag; Conotoxins; Sodium channels; disulphide rich peptides
