The auxiliary subunits Neto1 and Neto2 have distinct, subunit-dependent effects at recombinant GluK1- and GluK2-containing kainate receptors

Abstract

The kainate-type of ionotropic glutamate receptors are assembled from a combination of five different pore-forming subunits (GluK1-5), which confer distinct functional and pharmacological properties. These receptors are also modulated by co-assembly with the auxiliary subunits Neto1 and Neto2. To determine the impact of variation in subunit composition on the functional interaction between kainate receptors and Neto subunits, the Neto subunits were combined with either GluK1 or GluK2 in HEK-293T cells and responses to glutamate examined through patch-clamp recordings. Co-expression of GluK1 with either Neto1 or Neto2 caused a substantial increase in glutamate sensitivity and a slowing of the onset of desensitization at low agonist concentrations. However, at higher glutamate concentrations the primary effect of Neto2 was to slow the onset of desensitization, while that of Neto1 was to increase recovery from desensitization. In contrast, co-expression of Neto2 with GluK2 homomeric receptors had only modest effects on glutamate sensitivity, but increased the rate of recovery from desensitization as well as slowing its onset at all agonist concentrations. The properties of chimeric Neto1/Neto2 subunits suggested that the extracellular N-terminal region including the two CUB domains was largely responsible for the distinct regulatory effects of Neto1 and Neto2 on the desensitization properties of GluK1 homomeric receptors. These results further demonstrate that the functional effects of Neto subunits depend upon the subunit identity of both the auxiliary and the pore-forming subunits.

Neto Auxiliary Subunits Regulate Interneuron Somatodendritic and Presynaptic Kainate Receptors to Control Network Inhibition

“…Regulation of interneuron somatodendritic GluK2-dominated KARs by Neto1 is entirely consistent with findings in CA3 pyramidal cells where Neto1 controls somatodendritic/synaptic GluK2/5 heteromeric KARs (Straub et al, 2011a; Tang et al, 2011; Wyeth et al, 2014). Neto1 regulation of native GluK1-containing receptors on interneurons is similarly consistent with clear evidence for Neto1 association with and regulation of recombinant GluK1-containing KARs and GluK1-containing receptors at immature glutamatergic synapses (Copits et al, 2011; Fisher, 2015; Fisher and Mott, 2013; Orav et al, 2017; Palacios-Filardo et al, 2016; Sheng et al, 2015). The KARs regulating CCK/CB1 presynaptic release are dominated by GluK1 (Daw et al, 2010; Bureau et al, 1998; Christensen et al, 2004).…”

“…Despite overwhelming evidence supporting the ability of Neto2 to regulate recombinant GluK1-, GluK2-, and GluK5-containing KARs (Copits et al, 2011; Fisher, 2015; Fisher and Mott, 2012, 2013; Griffith and Swanson, 2015; Palacios-Filardo et al, 2016; Sheng et al, 2015; Sheng et al, 2017; Straub et al, 2011b; Zhang et al, 2009) and native GluK1 containing KARs in developing peripheral neurons (Vernon and Swanson, 2017), physiological relevance for endogenous Neto2 functional regulation of native KARs in central neurons has remained elusive. Indeed prior work failed to detect any defect in CA3 pyramidal cell KAR function in Neto2KO mice and Neto2 was not able to compensate for the dramatic defects observed in Neto1KOs (Tang et al, 2011, Wyeth et al, 2014, Straub et al, 2011a).…”

“…In recombinant studies Neto association with GluK1 increases glutamate sensitivity by 10–30 fold, greatly reduces macroscopic desensitization in response to submaximal (both Neto1 and 2)/maximal (Neto2) glutamate concentrations, and speeds recovery from desensitization (Fisher, 2015; Palacios-Filardo et al, 2015; Copits and Swanson, 2011; Straub et al, 2011b). Combined these functional changes could promote tonic activation of GluK1 KARs by low ambient glutamate levels (Fisher, 2015).…”

“…Recently, Neto1/Neto2 have emerged as auxiliary KAR subunits capable of regulating almost every parameter of receptor function (Copits and Swanson, 2012; Howe, 2015). Overexpression studies in heterologous cells or neurons have demonstrated that Netos regulate KAR desensitization and deactivation kinetics, channel open probability, ligand affinity, ion permeation, and subcellular localization (Brown et al, 2016; Copits et al, 2011; Fisher, 2015; Fisher and Mott, 2012, 2013; Griffith and Swanson, 2015; Orav et al, 2017; Palacios-Filardo et al, 2016; Zhang et al, 2014; Zhang et al, 2009). Consistent with these findings, studies at hippocampal mossy fiber to CA3 pyramidal cell (MF-CA3) synapses indicate that Neto1 regulates binding affinity, kinetics, and synaptic targeting of native GluK2/3-containing postsynaptic KARs (Straub et al, 2011a; Tang et al, 2011; Wyeth et al, 2014).…”

“…Importantly, Neto-mediated regulation of recombinant KARs can exhibit GluK subunit and Neto isoform specificity (Copits et al, 2011; Fisher, 2015). Thus, as Neto1/2 and GluK1-5 display discrete expression profiles throughout the CNS it is critical to consider network and cell-type specificity in Neto regulation of native KARs.…”