Modulation of the dimer interface at ionotropic glutamate-like receptor delta2 by D-serine and extracellular calcium - PubMed (original) (raw)

A, Side and top view of the dimer formed by two agonist-binding domains of GluRδ2 in the absence of

-serine (apo_-form) (PDB code 2V3T) (Naur et al., 2007). Soluble proteins containing the isolated agonist-binding domain of iGluRs have been constructed by deleting the N-terminal domain and replacing the transmembrane domains with a short linker. In these structures, the agonist-binding domain exists in a bilobed clamshell-like arrangement with the agonist-binding pocket located deep within the cleft between two lobes (D1 and D2). The two GluRδ2 subunits (colored yellow and orange) form a dimer that is symmetrical around a pseudo twofold axis. The dimer binds two calcium ions (shown as green spheres) at the top of the dimer interface. Residues at the dimer interface that were mutated in this study are shown as space-filled residues. B, Magnified top view of the solvent-accessible Ca2+-binding site with the backbone of one agonist-binding domain (protomer) shown in yellow and the other shown in orange. Dashed lines indicate potential coordination of Ca2+ by residues in the GluRδ2 dimer interface. The Ca2+-binding site is ideally situated to modulate the stability of the dimer as it is formed by acidic residues from both subunits. More specifically, Ca2+ interacts with E531 (backbone carbonyl and sidechain carboxyl), V534 (backbone carbonyl), and D535 (sidechain carboxyl) from one subunit and D782 (sidechain carboxyl) from the other subunit. The “top” of the Ca2+-binding site (i.e., the opposite side of Ca2+ relative to the sidechain carboxyl of E531) is exposed to the solvent. Residues that were mutated in this study and V534 are shown as gray sticks. The following two pairs of solvent-accessible cysteine mutations were made in GluRδ2Lc: E531C+D782C and D535C+D782C. Residues P528 and L789, which do not participate directly in Ca2+ binding, were also mutated to cysteines (Fig. 5_A). C–F, Concentration–response data for

-serine at GluRδ2Lc (C) and the GluRδ2Lc mutations D782A (D), D535A (E), and E531A (F) in the absence (circles) and presence (triangles) of extracellular Ca2+ (3 m

). EC50 values are listed in Table 1.

-Serine EC50 (i.e., potency) at GluRδ2Lc is highly sensitive to extracellular Ca2+, whereas

-serine EC50 at GluRδ2Lc D535A and D782A is unaffected by extracellular Ca2+. The EC50 of

-serine at GluRδ2Lc E531A is less sensitive to extracellular Ca2+. G, Summary of fold shifts in

-serine EC50 in the presence of extracellular Ca2+ (3 m

) relative to

-serine EC50 in the absence of extracellular Ca2+. H, Summary of fold shifts (i.e., Ca2+ potentiation) of spontaneously active currents in the presence of extracellular Ca2+ (3 m

) relative to baseline spontaneously active currents in the absence of extracellular Ca2+. Numerical values are listed in Table 1. Black bars indicate fold shift at nonmutated GluRδ2Lc, white bars indicate GluRδ2Lc with mutations in the

-serine-binding site, striped bars indicate GluRδ2Lc with mutations in the Ca2+-binding site, and n.d. indicates not determined. Fold shifts of

-serine EC50 were not determined at R530K and D742A, since

-serine EC50 could not be determined at these mutants.