| Kainate receptors are members of the ionotropic glutamate receptor family that modulate neurotransmitter release and intrinsic neuronal excitability. Consequently, unlike AMPA receptors and NMDA receptors, they primarily serve a modulatory role in nervous system function. Moreover, their activity is shaped further by incorporation of the auxiliary proteins Neto1 and Neto2, which changes every parameter of receptor function examined to date, including rates of desensitization, deactivation, and open probability. However, the structural basis of this modulation remains unknown.;Herein, we identified two regions within kainate receptors, the M3-S2 linkers and the D1 dimer interface, that are critical for functional modulation of desensitization by the auxiliary subunit Neto2. Mutations in the M3-S2 gating linker had bidirectional effects on both basal receptor desensitization and Neto2 modulation; however we did not find a correlation between these two parameters. That is, some mutations that impacted receptor desensitization had no effect on Neto2 modulation and conversely, we identified mutations that did not impact receptor desensitization but significantly altered Neto2 modulation. While the M3-S2 linkers influence the magnitude and direction of Neto2 modulation, we found that the integrity of the ligand binding domain dimer interface is required for Neto2 modulation of desensitization. Additionally, using cation sensitivity as an indirect readout, we determined that association with Neto2 stabilizes this dimer interface, priming receptors for activation. Finally, using chimeric kainate receptor constructs we attempted to identify regions within the receptor that mediate the subunit-specificity of Neto2 modulation of desensitization. Although we were unable to confirm the regions and residues underlying this specificity, we were able to exclude the amino terminal domain, the dimer interface, and the intracellular carboxy-terminus. Furthermore, experiments with Neto protein chimeras confirmed functional modulation occurs via the extracellular domain and engages different regions within Neto1 and Neto2 dependent upon the receptor subunit composition. This is the first study to identify regions within kainate receptors targeted for modulation by auxiliary proteins. Auxiliary subunits act as allosteric modulators of receptor function; hence our work may guide the rational development of KAR-specific pharmacological tools. |
