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Jansen Laboratory - Research

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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).

Receptor: Ligand: Clinically targeted for:
nAChR Acetylcholine Treatment of Nicotine addiction, Alzheimer's Disease
GABAA gamma-Aminobutyric acid Treatment of Epilepsy, Anaesthesia, Muscle relaxation, Anxiolytics
5-HT3A 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.
Subunit Topology 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 (TEVC).
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).
Disulfide Crosslinking 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.

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