Identification of regulatory proteins of Acid-Sensing Ion Channels

Acid-sensing ion channels (ASICs) are voltage-independent proton-gated ion channels belonging to the amiloride-sensitive degenerin/epithelial Na+ channel (DEG/ENaC) family o f receptor channels. Six subunits (A SICla, lb, 2a, 2b, 3 and 4) encoded for by four genes, have been identified so far. All ASIC subunits are expressed in dorsal root ganglia (DRG) and have been implicated in physiological sensory processes such as nociception associated with tissue acidosis, cutaneous and visceral mechanosensation, sour taste and cochlear function. However, some ASIC subunits also show a wide distribution throughout the brain, where they are thought to modulate synaptic communication. Supporting this hypothesis several studies demonstrated in mice a role for ASICs in learning, memory and fear behaviour. Recently ASIC la channels were also shown to make a major contribution to hippocampal neuronal damage in stroke through Ca2+ overload. ASIC4 is broadly expressed in the nervous system but is not gated by protons and has no known function. This thesis describes a genetic analysis carried out to identify interacting partners of ASICs in sensory neurons, in order to shed light on the possible role o f ASIC4, and to better understand the functional roles o f ASIC 1-3 and their regulatory mechanisms. A rat dorsal root ganglion cDNA library was screened in a yeast two-hybrid assay and a number o f proteins interacting with the N-terminal domain o f rat ASIC 1-4 were trapped. Many o f these proteins were involved in trafficking o f ion channels, G protein pathways, endocytosis, protein ubiquitination, or cell adhesion, suggesting potentially novel roles and regulatory mechanisms for ASIC channels. The annexin II light chain p l l was found to specifically interact with ASIC la, and to promote its functional expression at the plasma membrane, as shown by immunocytochemistry, cell surface protein biotinylation and electrophysiology. ASIC4 was shown to decrease the protein level o f other ASIC subunits, and to downregulate ASIC la-mediated currents. Preliminary data suggest that this effect may be due to an involvement o f ASIC4 in ubiquitination pathways. Overall, this work has led to the identification o f interacting proteins that regulate ASICs and suggested novel functions for ASIC4.