NMDA receptors display a characteristic inhibition by extracellular Mg2+ (Nowak et al., 1984). Addition of 2 mM Mg2+ had, however, no effect on IR84a+IR8a or IR75a+IR8a

currents measured when the primary charge carrier was Na+ (Figure S4A). We also tested several iGluR antagonists for their influence on IR-dependent currents, including two NMDA pore blockers, memantine and MK-801 (Kashiwagi et al., 2002), and an AMPA and Kainate receptor blocker, philanthotoxin (Jones et al., 1990 and Ragsdale et al., 1989). None of these had effects on either IR84a+IR8a or IR75a+IR8a currents, except for memantine, which inhibited phenylacetaldehyde-induced IR84a+IR8a currents with a half maximal inhibitory concentration (IC50) of 39 ± 9 μM (Figure S4B), a value that is ∼40 times the IC50 of memantine for NMDA receptors (Parsons et al., 2008). Antagonists for several other classes of ion channel, including amiloride, Cd2+, tetraethylammonium (TEA), selleck kinase inhibitor and ruthenium red, had mostly modest effects on IR84a+IR8a or IR75a+IR8a currents, even at high concentrations (Figure S4B). Notably, while ruthenium red slightly inhibited IR84a+IR8a currents, it enhanced IR75a+IR8a current

amplitudes (Figure S4B). Together, these experiments distinguish IRs pharmacologically from both iGluRs and other classes of ion channel, and further highlight the physiological differences between different IR complexes. To understand the molecular basis for the functional heterogeneity JAK inhibitor of IR84a+IR8a and IR75a+IR8a, we compared the sequence of the putative ion channel pore domains of IR84a, IR75a, and IR8a with those of iGluRs. While this region is highly conserved in iGluRs, individual IRs bear a large number of amino acid substitutions (Figure 6B). This sequence divergence may account for the observed insensitivity of IRs to iGluR pore blockers as well as the pharmacological differences between IR84a+IR8a and IR75a+IR8a (Figures 6A, S4A, and S4B). We focused on residues aligned with a glutamine that controls Ca2+ permeability in iGluRs (Dingledine et al., 1992) (Figure 6B). In GluA2, RNA editing-regulated

substitution of this glutamine to arginine renders channels Ca2+-impermeable of (Hume et al., 1991 and Liu and Zukin, 2007). While IR75a contains an isoleucine (I388) in this position, IR84a retains a glutamine (Q401) (Figure 6B). We hypothesized that this residue might account for the difference in Ca2+ conductance mediated by IR75a+IR8a and IR84a+IR8a channels (Figure 6A). To test this, we generated an IR84aQ401R mutant receptor, which we predicted to lack Ca2+ permeability. IR84aQ401R+IR8a expressing oocytes showed similar Na+ current amplitudes (Figure 6C) and phenylacetaldehyde concentration responses as the wild-type receptors (Figure 6D). Importantly, IV curve measurements revealed that IR84aQ401R+IR8a-dependent conductance of monovalent cations was unchanged compared with the wild-type receptors, but that Ca2+-dependent conductance was abolished (Figure 6E).