A Nondesensitizing Kainate Receptor Point Mutant

Abstract

Ionotropic glutamate receptor (iGluR) desensitization can be modulated by mutations that change the stability of a dimer formed by the agonist binding domain. Desensitization of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be blocked by a single point mutation (e.g., GluR2 L483Y) that stabilizes this dimer in an active conformation. In contrast, desensitization of kainate receptors can be slowed, but not blocked, by similar dimer interface mutations. Only covalent cross-linking via introduced disulfides has been previously shown to block kainate receptor desensitization completely. We have now identified an apparently nondesensitizing GluR6 point mutant (D776K) located at the apex of the ligand binding (S1S2) domain dimer interface. Asp776 is one of a cluster of four charged residues in this region that together mediate direct dimer interactions and contribute to the binding sites for one chloride and two sodium ions. Despite the localized ϩ4 change in the net charge of the S1S2 dimer, the D776K mutation actually increased the thermodynamic stability of the dimer. Unlike GluR6 wild type, the D776K mutant is insensitive to external cations but retains sensitivity to external anions. We therefore hypothesize that the unexpected phenotype of this charge reversal mutation results from the substitution of the sodium ions bound within the dimer interface by the introduced lysine NH 3 ϩ groups. The nondesensitizing D776K mutant provides insights into kainate receptor gating and represents a potentially useful new tool for dissecting kainate receptor function.

Conformational Flexibility of the Ligand-Binding Domain Dimer in Kainate Receptor Gating and Desensitization

“…Construction, mutagenesis and expression of the GluK2 pET21a plasmid was as described previously (Nayeem et al, 2009) Studier, 2005) for 20 h at 27°C, and terminal tags were removed before gel filtration by thrombin digestion. Constructs therefore comprised Ser429-Lys544 (the S1 domain), a GlyThr linker and Pro667-Glu806 (the S2 domain), plus an N-terminal Gly and C-terminal ProArg from the thrombin sites.…”

“…The M770K and D776K mutations differ in their effects on receptor sensitivity to external anions, with responses unaffected by exchange of anions in GluK2-M770K, but attenuated in GluK2-D776K (Wong et al, 2007;Nayeem et al, 2009). The anionbinding site in kainate subunits is located within the dimer interface, lying between two pairs of salt bridges formed between Arg775-Asp776 and Glu524-Lys531 (Plested and Mayer, 2007).…”

“…Stabilization of the D1-dimer therefore attenuates desensitization (Sun et al, 2002;Chaudhry et al, 2009b). Macroscopic desensitization can be blocked by covalent cross-links (Priel et al, 2006;Weston et al, 2006) and single point-mutations (Stern-Bach et al, 1998;Nayeem et al, 2009) in both AMPA and kainate receptors. In AMPA subunits a leucine-to-tyrosine mutation increases dimer affinity by Ͼ10 5 -fold through increased contacts between D1 domains (Sun et al, 2002).…”

“…In AMPA subunits a leucine-to-tyrosine mutation increases dimer affinity by Ͼ10 5 -fold through increased contacts between D1 domains (Sun et al, 2002). Macroscopic desensitization is only slowed when these interactions are recreated in kainate subunits (Weston et al, 2006;Zhang et al, 2006), but it is blocked by the charge-reversal mutation GluK2-D776K (Nayeem et al, 2009). This residue is one of a cluster of four charged-residues involved in inter-dimer contacts at the LBD apex (Fig.…”

“…We hypothesized that the GluK2-D776K phenotype results from sodium-ion displacement by the introduced lysine, because unlike wild-type (WT), GluK2-D776K is unaffected by replacement of extracellular sodium by other cations (Nayeem et al, 2009). Here we have used x-ray crystallography to determine structural models for GluK2-D776K in complex with glutamate (Glu) and KA, along with GluK2-WT and a second lysine mutant, GluK2-M770K.…”