Creation and optimization of a genetically encoded sensor for studying the serotonergic system
Date Issued
2023-02-15
Author(s)
Supervisors
Abstract
In this work, a genetically encoded fluorescent biosensor for the detection of serotonin was produced. The sensor consists of two proteins, cpGFP and the 5-HT1A receptor, a GPCR involved in the transmission of extracellular signals into the intracellular lumen across the plasma membrane. Ultimately, the substitution of a large part of the third intracellular loop of the 5-HT1A receptor with cpGFP as well as various mutations in specific linker domains between the two proteins, led to the genetically encoded serotonin sensor sDarken.
The sensor sDarken can detect serotonin by a strong reduction of its fluorescence after binding of serotonin. Although not predominantly expressed in the membrane, the fluorescence was most intense in the membrane portions of HEK cells where sDarken was expressed. The sensor sDarken can detect multiple sequential applications of serotonin and showed no changes in its fluorescence response, while the cells were embedded within media with different pH values. Furthermore, other chemicals failed to induce a similar reduction in fluorescence, with the exception of the 5-HT1A receptor agonist 8-OH-DPAT as well as the 5-HT1A receptor antagonist WAY-100635, suggesting a specificity of the sensor for serotonin.
Measurement of the affinity to serotonin show that sDarken is capable to measure serotonin within physiological relevant concentrations.
The sensor sDarken can detect serotonin by a strong reduction of its fluorescence after binding of serotonin. Although not predominantly expressed in the membrane, the fluorescence was most intense in the membrane portions of HEK cells where sDarken was expressed. The sensor sDarken can detect multiple sequential applications of serotonin and showed no changes in its fluorescence response, while the cells were embedded within media with different pH values. Furthermore, other chemicals failed to induce a similar reduction in fluorescence, with the exception of the 5-HT1A receptor agonist 8-OH-DPAT as well as the 5-HT1A receptor antagonist WAY-100635, suggesting a specificity of the sensor for serotonin.
Measurement of the affinity to serotonin show that sDarken is capable to measure serotonin within physiological relevant concentrations.
Subjects
genetical encoded sensor
;
serotonin
;
GPCR
Institution
Type
Dissertation
Language
English
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Dissertation Martin Kubitschke.pdf
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44.42 MB
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