Jansen Laboratory - Research
Structure and function studies of ligand-gated ion channels and transporters
Michaela Jansen's primary research focus are structure-function studies of membrane proteins on the molecular level. Methods used include a wide array of biophysical (X-ray crystallography, electrophysiology),
molecular biology (protein engineering) and biochemical approaches.
As heterologous expression hosts we mainly use E. coli, HEK, CHO, and Xenopus laevis oocytes.
One main focus are the so called Cys Loop receptors. These receptors function as neurotransmitter
receptors. They transform the chemical signal contained in the neurotransmitter into
an electrical signal. The superfamily of Cys Loop receptors has a variety of members,
that are named after the ligand that opens (gates) them.
These receptors were also identified in prokaryotes and we were the first lab to engineer
functional chimeras by adding the intracellular domain that is only found in eukaryotic receptors to
the proton-gated channel from Gloeobacter violaceus (GLIC).
||Clinically targeted for:
||Treatment of Nicotine addiction, Alzheimer's Disease
||Treatment of Epilepsy, Anaesthesia, Muscle relaxation, Anxiolytics
||5-hydroxytryptamine = serotonine
||Treatment of Depression, Nausea in Chemotherapy
Other members of the superfamily include receptors for glycine, zinc, glutamate, and
protons. Each receptor is made up of five subunits that are arranged pseudosymmetrically
around the central ion-conducting pore. These subunits can change their conformation
from closed (non ion conducting) to open (ion conducting) states.
||The overall topology of the individual subunits is comparable. The ~200 amino acid
long extracellular domain (ECD) harbors the ligand binding domain and the eponymous
13 amino acids bridging disulfide loop. Four alpha-helical segments (M1-M4) form the
transmembrane domain (TMD). Two short loops, one intracellular between M1 and M2 and
one extracellular between M2 and M3 connect the transmembrane domains. The large M3M4
loop is the major contributor to the intracellular domain (ICD). The intracellular
domain is reduced to a minimum in prokaryotic Cys Loop Receptors. We have shown that
the long M3M4 loop in 5HT3A and GABA rho1 can be replaced by just a heptapeptide linker found in GLIC while retaining
functionality. We have also generated the opposite chimeras between ECD and TMD of
GLIC and diverse eukrayotic ICDs.
||Homology Models of Eukaryotic and prokaryotic-like 5HT-3A receptors
||The structure and function of these receptors is studied in wildtype or engineered
receptors. Very often we engineer receptors by using site-directed mutagenesis to
selectively change one amino acid to a cysteine. In-vitro mRNA is synthesized by standard
molecular biological methods, injected into Xenopus laevis oocytes, and the function investigated in Two-electrode voltage clamp experiments
|Two-Electrode Voltage Clamp
||Setup for TEVC
||The substituted cysteine accessibility method (SCAM) is utilized to elucidate the
structure. This method was pioneered by Myles H. Akabas and Arthur Karlin. It can
be used to investigate the water accessible amino acid positions of a certain protein,
and it is also applied for studies of the steric and electrostatic micro-environments,
and changes to these micro-environments during the opening of the channel.
|MTS-Reagent Modification of Cysteine
||Another technique routinely used to investigate proximity relationships is disulfide
crosslinking, two engineered cysteines are cross-linked to form a cystine that contains
two disulfide linked cysteines either spontaneously or by applying an oxidizing reagent.
This reaction can be monitored with the two electrode voltage clamp technique if it
occurs inside the same subunit (intra subunit) or between different subunits (inter
subunit) and also by Western blotting if the cross-link is between two different subunits
(inter-subunit). Most disulfide bond formations can be reversed by applying a suitable
reducing agent (DTT, TCEP).
||Homology Model used for Crosslinking Study
Michaela Jansen also uses other biophysical, protein engineering and biochemical approaches
to investigate these receptors.
Homology modeling of proteins is used to design new projects, visualize and explain
results, and for preparing figures for publications!
Ligands for Ion-Channels
Michaela Jansen is also interested in the development of glycine-site NMDA antagonists
(QSAR, ligands for PET), as well as the development of subtype selective GABAA receptor ligands.