Functional characterization of the ASIC1a proteome

• Applicant: Professor Dr. Stefan Gründer
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 539767547

Acid-sensing ion channels are proton-gated Na+ channels with broad distribution in the central and peripheral nervous system. ASICs are trimers, and ASIC1a is the principal ASIC subunit in the CNS, which is included in almost all homo- and heterotrimeric ASICs. ASIC1a localizes to dendritic spines and contributes to excitatory postsynaptic currents at excitatory synapses. It promotes long-term potentiation. In addition to its role in synaptic transmission, ASIC1a senses sustained acidosis during pathophysiological states such as ischemic stroke. Accordingly, the activation of ASIC1a during ischemic stroke leads to enhanced neuronal death in animal models. After heterologous expression, ASIC1a mostly localizes to intracellular organelles, mostly the endoplasmic reticulum, and it is unknown, which factors control the expression of ASIC1a at the surface of neurons and in the postsynaptic membrane. To characterize the ASIC1a proteome, we commissioned an affinity purification-mass spectrometry (AP-MS) screen of native ASIC1a channels from the mouse brain. Among a few other proteins, this screen identified an uncharacterized protein with unknown function as a specific partner of ASIC1a. In preliminary experiments, we could show that this protein is secreted, why we call it secreted protein 1 (SEP1). SEP1 increases the current amplitude and surface expression of ASIC1a. On the other hand, SEP1 localizes to the surface of cells that also express ASIC1a. Strikingly, in neurons from mice with knockout of the SEP1 gene, ASIC1a currents were completely absent, suggesting an essential role of SEP1 in the function of ASIC1a. In this grant application, I propose 1) to characterize the molecular mechanism of the increased current amplitude and surface expression of ASIC1a by SEP1, 2) to further characterize the interaction of the two proteins in primary neurons, 3) to characterize the molecular properties of SEP1, 4) to reveal the molecular structure of the ASIC1a-SEP1 complex, and 5) to investigate the influence of SEP1 on the neurodegenerative potential of ASIC1a. To achieve our goals, we will use a multitude of molecular, biochemical, cell biological and electrophysiological methods, including fluorescence confocal microscopy, patch clamp recordings, recordings on acute brain slices, immunohistochemistry, viral transfection, subcellular fractionation, siRNAs, and (in collaboration) cryo-EM; in addition, we have established a colony of mice with a genetic knock-out of the new ASIC1a auxiliary subunit. Together, the application will reveal the influence of SEP1 on trafficking, subcellular localization, and electrophysiological properties of ASIC1a, as well as on neuronal excitability.

• DFG Programme: Research Grants
• International Connection: USA
• Co-Investigator: Professor Dr. Nikolaus Plesnila
• Cooperation Partner: Isabelle Baconguis, Ph.D.