Prof. Dr. Michael Hollmann, Biochemistry I - Receptor Biochemistry, Faculty of Chemistry and Biochemistry
IGSN Students supervised to date: Zhan Lu Ma (admitted Oct 2002), Sabine Schmidt (admitted Oct 2003), Elke Muth-Köhne (admitted Oct 2005), Nora Cavara (admitted Oct 2006)
Molecular basis of glutamatergic synaptic transmission
Structure, function, regulation, and modulation of ionotropic glutamate receptors is investigated by molecular biological, electrophysiological, proteinbiochemical and immunocytochemical procedures. We analyze structural and functional properties of members of the extended protein family of ionotropic glutamate receptors in heterologous expression systems such as Xenopus laevis oocytes and HEK293 cells. In our studies we include vertebrates glutamate receptors (23 known genes) as well as invertebrate and plant glutamate receptors such as those of Caenorhabditis elegans (15 genes) and Arabidopsis thaliana (20 genes), respectively.
Current projects of IGSN students:
Mechanisms of glutamate receptor assembly, and rules governing the assembly of heteromeric glutamate receptors
Ionotropic glutamate receptors are heterotetrameric protein complexes that in mammals are formed from a total of 18 subunits which can be classified in 4 subfamilies. Members of different subfamilies appear not to cointegrate into the same receptor complex, while members of the same subfamily are promiscuous. We use fluorescent protein-fused glutamate receptor subunits and confocal microscopy to investigate the process of assembly during glutamate receptor expression in Xenopus oocytes and HEK293 cells. Different varieties of the fluorescent protein GFP, such as EGFP, EYFP, and ECFP as well as DsRed2 are used to engineer distinctly labeled receptor subunits to monitor the formation of heteromeric receptor complexes by colocalization analysis as well as FRET studies. Mutagenesis of domains suspected of controlling the assembly process is used to test hypotheses of regulatory mechanisms.
Characterization of functional domains of the orphan glutamate receptors delta1 and delta2
Two of the 18 mammalian glutamate receptor subunits, delta1 and delta2, have eluded functional characterization despite their
obvious sequence homologies with other glutamate receptor subunits. They neither form functional ion channels upon
expression in Xenopus oocytes or HEK cells, nor do they bind any of the known glutamatergic ligands.
The only available hint at function is the existence of a mutant mouse, the lurcher mouse, which suffers from ataxia and Purkinje cell atrophy in the cerebellum due to a point mutation in the delta2 gene.
To investigate why delta subunits are non-functional we use a domain transplantation approach. Domains known to carry distinct functions in glutamate receptors, such as the ligand binding domain, the ion pore domain, and the linker domains connecting the latter two domains are inserted into proven functional glutamate receptor subunits such as the AMPA receptor subunit GluR1 or the kainate receptor subunit GluR6 and tested for function in their new environment. With this approach we hope to be able to pinpoint the functionally critical domains in delta receptor subunits, and at the same time gain insight into possible gating domains of the glutamate receptor family.
Glutamate receptors during mouse stem cell differentiation
Omnipotent embryonal stem cells (ES cells) can be cultured and differentiated into neural progenitor cells which eventually will terminally differentiate into glial or neuronal cells. We would like to elucidate if glutamate receptors play a role in this differentiation process, or whether their expression patterns are shaped by differentiation. In addition, we would like to determine how early on during cell differentiation the various mammalian glutamate receptor subunits are expressed. To address these questions we analyze glutamate receptor expression by real-time RT-PCR in an engineered mouse ES cell line (46C cells) which expresses green fluorescent protein off the sox1 promoter. This fluorescent marker allows for selection of sox1-expressing (“green”) neural progenitor cells which then can be analyzed separately. Finally, progenitor cells are differentiated into glial and neuronal cells which are also analyzed for their glutamate receptor expression patterns. This approach will allow us to gauge potential roles the various glutamate receptors may play during differentiation, and it may identify key subunits which could then be knocked down/out to rigorously prove their importance in cellular development.
Investigation of the functional relevance of the NR3A and NR3B subunits of NMDA receptors
The most complex and most highly modulated and regulated member of the ionotropic GluR family is the N-methyl-D-aspartate receptor (NMDAR). NMDA receptor subunits assemble into obligatory heteromers, which need glycine (or D-serine) in addition to glutamate as an essential coagonist. They are highly calcium-permeable and susceptible to inhibition by proteins, amines, and to blockage by Mg2+ ions. NMDAR function at the molecular level has been well studied with regard to the NR1 and NR2 subunits, but knowledge about the role of the NR3 subunits is scant. Expression of the two NR3 subunits is regionally and developmentally restricted. It is generally believed that both NR3 subunits have a merely modulatory role, serving as dominant-negative regulators of NR1/NR2 heteromers with regard to current amplitude and Ca2+ permeability. This may serve to lower NMDAR excitability, and, consequently, to protect neurons selectively from glutamate-induced excitotoxicity.
Previous studies to elucidate the function of NR3 have focused solely on a very limited subset of subunit combinations, taking into account primarily the interaction of NR3 with a single splice variant of NR1 (out of eight) and one (NR2A) out of four NR2 subunits. We are investigating the interactions of NR3B with all eight functional NR1 splice variants and all four NR2 subunits. Co-expression analyses in a heterologous expression system using Xenopus laevis oocytes have already revealed a far more differentiated influence of NR3 on NMDAR function than the widely assumed generally inhibitory modulatory effect. Another aspect of this project is a fresh look at the reported glutamate-insensitive NMDA receptor subunit combination NR1/NR3 which has been suggested to be in effect an excitatory glycine receptor.