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

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.