Functional Reconstitution of SLC18 Family Transporters

by Abhishek Acharya
Doctoral thesis
Date of Examination:2024-12-18
Date of issue:2025-12-12
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Holger Stark
Persistent Address: http://dx.doi.org/10.53846/goediss-11685

 

 

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Abstract

English

The Solute Carrier (SLC) family proteins are a diverse group of proteins performing many vital functions from bacteria to humans. Vesicular neurotransmitter transporters (VNTs) also belong to the SLC family. Based on amino acid sequence, substrate specificity and transport mechanism, they are classified into three different families namely SLC17, SLC18 and SLC32. They mediate storage of neurotransmitter molecules against concentration gradient and are driven by either a proton or an electrochemical gradient. SLC18 family is a group of VNTs that function to transport positively charged monoamines inside synaptic vesicles, consisting of vesicular monoamine transporters (VMATs) 1 and 2, vesicular acetylcholine transporter VAChT and the vesicular polyamine transporter (VPAT). These proteins are involved in many pathways central for life due to the neurotransmitters they transport and are very important drug targets. Existing in vivo studies show these transporters act as antiporters, exchanging two protons against one substrate molecule with the proton gradient provided by the vacuolar ATPase. Structural and biochemical characterization of these transporters is a perquisite to advance our understanding of their molecularfunctioning, regulation and possible pharmacological targeting. Until recently, high resolution 3D structure and molecular mechanism of transport for these family proteins was unknown. This study aims at the purification, reconstitution and transport studies with SLC18 transporters, as well as sample preparation with full length VAChT for cryo electron microscopy studies. In this work a robust and reproducible protocol for purifying full length VMAT2 and VAChT, as well as fluorescent sensing fluorescent reporters is developed. Binding assays using VMAT2 show a binding with the inhibitor reserpine were established. Transport assays using fluorescent substrates for VMAT2 (FFN) and acetylcholine sensing fluorescent reporter (iAChSnFR) were established. The results also show that the transport can be inhibited using transporter specific inhibitors reserpine and vesamicol, as well as by inhibiting the co-reconstituted ATPase, which works as a proton pump. This shows the functional reconstitution of the SLC18 transporters and their dependence on a proton gradient.
Keywords: SLC18; VNTs; neurotransmitter; transporters
 
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