Energetical contributions of intersubunit salt bridges of rASIC1a and investigations about the stoichiometry of rASIC1a/2a

Acid-sensing ion channels (ASICs) are proton gated cation channels, which are involvedin many neuronal diseases, synaptic transmission, learning, memory and nociception.ASICs are members of the DEG/ENaC gene family and consist of four genes (ASIC1-4),with two splice variants (ASIC1b and 2b). They are mainly expressed in the central andperipheral nervous system. Their implication in neuronal diseases makes them a potentdrug target. The knowledge of their gating mechanism and the exact composition ofASIC in heteromers are the basis for the development of new therapeutic ligands forthe treatment of disease patterns, where ASICs are involved.This study can be divided into two parts. First, the interaction during the gatingof rASIC1a between two conserved histidines and an aspartate was investigated inmolecular detail and second, the exact stoichiometry of the ASIC1a/2a heteromer hasbeen identified.For the investigation of the gating mechanism, previous studies gave first insightsinto the functional relevance of specific amino acids. Studies on rASIC1a found thatthe double mutation of the fully conserved H73 and the partially conserved H72into asparagine led to non functional channels. Additionally, if D78 was mutatedinto asparagine together with mutations in the Ca2+ binding site E425GD432C, thechannel function was abolished. These results indicate a relevance of these aminoacids for proper channel gating. Furthermore, the crystal structure of cASIC1 inthe desensitized state gave first hints for an interaction between H73 and D78 of anadjacent subunit by building a salt bridge.By the method of the double-mutant cycle analysis, the formation of a salt bridgebetween H72 and H73 to D78 of an adjacent subunit and its energetical contributionto the different conformations of the channel was explored. Furthermore, H72, H73and D78 were mutated into cysteines and their spatial proximity was investigated bydisulfide bond trapping. Additionally, the application of MTS reagents and the chargeswap and reversal of the amino acids gave information about the importance of thecharge of the respective side chains and their structural rearrangement during gating.The results of this study suggest stabilizing contributions of the salt bridge betweenH73 and D78 to ligand binding and of the interaction between H72 and D78 todesensitization. During gating, the channel experiences a structural rearrangementabove the transmembrane domains, which is supported by the energetical contributionsof the salt bridges. The freely moving side chain of D78 and the positive chargeseither at position H72 or at H73 are essential for proper channel function. In summary,not just the salt bridges but the structural rearrangement above the transmembranedomain around D78 influence the channel during pore opening.In the second part of the thesis, the stoichiometry of ASIC1a/2a heteromers hadbeen explored. ASIC1a/2a heteromers had been described for example in hippocampaland dorsal horn neurons, where they show distinct functional properties from theirrelative homomers. Since drugs often bind at subunit interfaces and different agonistsand antagonists of ASICs have been found by crystal analysis to bind at interfaces, theknowledge of the subunit composition in heteromers has a relevance for drug design.For the electrophysiological characterization of the heteromers, the method of thetwo-electrode voltage-clamp was used. Furthermore, the group of Prof. Dr. Ulbrichfrom Freiburg counted the GFP and ttCherry tagged ASIC subunits in the heteromerby bleaching steps with a single molecule imaging method.The results of the second part clearly revealed the coexistence of ASIC1a andASIC2a homomers, together with ASIC1a/2a heteromers in Xenopus laevis oocytes.Heteromeric ASIC1a/2a assembles in a random fashion, with either a 1:2 or a 2:1stoichiometry. This flexible stoichiometry of ASIC1a/2a increases the variety of ASICsin the central and peripheral nervous system