Role of Neto1 extracellular domain in modulation of kainate receptors.

Kainate receptors play fundamental roles in regulating synaptic transmission and plasticity in central nervous system and are regulated by their cognate auxiliary subunits Neuropilin and tolloid-like proteins 1 and 2 (Neto). While electrophysiology-based insights into functions of Neto proteins are known, biophysical and biochemical studies into Neto proteins have been largely missing till-date. Our biochemical, biophysical, and functional characterization of the purified extracellular domain (ECD) of Neto1 shows that Neto1-ECD exists as monomers in solution and has a micromolar affinity for GluK2 receptors in apo state or closed state. Remarkably, the affinity was ~2.8 fold lower for receptors trapped in the desensitized state, highlighting the conformation-dependent interaction of Neto proteins with kainate receptors. SAXS analysis of Neto1-ECD reveals that their dimensions are long enough to span the entire extracellular domain of kainate receptors. The shape and conformation of Neto1-ECD seems to be altered by calcium ions pointing towards its possible role in modulating Neto1 functions. Functional assays using GluK2 receptors and GluK2/GluA2 chimeric receptors reveal a differential role of Neto1 domains in modulating receptor functions. Although the desensitization rate was not affected by the Neto1-ECD, the recovery rates from the desensitized state are altered.