Background and previous work
Dorsal root ganglia (DRG) are the main relay stations between the periphery and the central nervous system, harbouring motoneurons and Aδ/C fibres essential for pain perception. Oxidative stress and a decay of the antioxidant system result in aberration of electrical signalling, particularly in aged organisms. This process appears to be linked to an accumulation of oxidised methionine residues (methionine sulfoxide). This project is based on previous work, in which we have characterised human methionine sulfoxide reductases (MSRs) and their role for degenerative diseases and selected ion channel function. Furthermore, our preliminary experiments have shown that voltage-gated sodium channels (NaV) expressed in DRG neurons (NaV1.7-NaV1.9) are particularly sensitive to oxidative modification.
Here we will elucidate how MSRs affect the electrical signalling in DRG neurons and how oxidative protein modification alters their voltage-gated sodium channels. We will address the following specific questions: (1) Which MSRs are expressed by the various cell types in DRGs and how do ageing and diet affect their expression and the status of methionine oxidation? (2) What are the functional consequences of methionine oxidation in DRGmediated electrical signalling? (3) What are the molecular mechanisms of age-dependent sodium channel modification? (4) Can we determine the status of protein modification with engineered fluorescence proteins?
Using methods of molecular biology and by means of electrophysiological recordings from DRGs of wild-type and transgenic mice (MSRA–/–, NaV1.8–/–, NaV1.9–/–) of varying age and diet, as well as by employing recombinant systems, we will address the above mentioned specific questions. The electrical signalling of DRG neurons will be assessed with whole cell current-clamp methods. The functional status and methionine oxidation dependence of select ion channels will be subjected to in-depth analysis in recombinant expression systems. Moreover, we will design and test various proteins based on green fluorescent protein (GFP) to assess the status of intracellular protein modification non-invasively using ratiometric fluorescence imaging. This study will contribute to a better understanding of how ageing and oxidative modification alter rapid electrical signalling and pain transmission. Our major collaborator is J. Balbach (SP8) for the structural elucidation of conformational changes induced by NaV channel protein modification. Further, collaborations are planned with SP1 and SP2 on protein glycation/sialylation, SP9 and SP13 on ROS signalling, and SP5 for protein analytics.