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Exploring solid supported membrane based electrophysiology as an alternative platform to probe activity of membrane transport proteins

Gantner, Matthias (2017) Exploring solid supported membrane based electrophysiology as an alternative platform to probe activity of membrane transport proteins. PhD thesis, University of Leeds.

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Membrane transport proteins take a pivotal role in all forms of life as they are responsible for organising traffic of ions and small molecules across the hydrophobic barrier of biological membranes. Mutations in membrane transporters can often lead to severe diseases and they often consitute drug targets. Hence, assaying function of membrane transporters is of great importance. In this project the method used for this task was mainly a relatively uncommon technique called solid-supported membrane based electrophysiology. The goal was to test this technique on targets that are challenging to investigate by more conventional methods. A first target was the TRPM2 ion channel. TRP channels are difficult to investigate because they often show a very complex activation pattern. A second target was the bacterial transition metal transporter MntH2 from Enterococcus faecalis, belonging to the SLC11 family. Transition metal transporters are generally difficult to investigate, because of the nature of their substrates. Some transition metals are redox-active and in solution they act as complexing agents. Application of solid supported membrane based electrophysiology was not successful for TRPM2, but the method was used to perform basic biophysi- II cal characterisation of MntH2. It was found that MntH2 transports a range of substrates including Mn2+, Cd2+,Co2+ and Zn2+. Ni2+ and Cu2+ were not transported and in fact inhibited manganese uptake. Interestingly, in the presence of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) electrophysiological currents were not affected. This, together with the observation from a complementary assay, that reconstituted MntH2 did not acidify the interior of vesicles loaded with pH-sensitive uorescence probes, led to the hypothesis that MntH2, contrary to common belief, is not a H+ symporter. MntH2 was attempted to crystallise and in initial screens some conditions were identified which could be a basis for optimisation in future trials.

Item Type: Thesis (PhD)
Keywords: "transition metals" "membrane transporter" "SLC11" "solid supported membrane based electrophysiology" "Enterococcus faecalis" "SURFE2R" "MntH2" "TRPM2" "proton coupling" "cotransport" "manganese"
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute of Membrane and Systems Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.737837
Depositing User: Mr Matthias Gantner
Date Deposited: 28 Mar 2018 14:06
Last Modified: 25 Jul 2018 09:56
URI: http://etheses.whiterose.ac.uk/id/eprint/19682

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