The glutamate receptor superfamily comprises a large number of different genes, each encoding a subunit that assembles to form the functional receptor complex, believed to be composed of four primary subunits (2). Glutamate receptors are traditionally divided into three classes, AMPA receptors, NMDA receptors and kainate receptors, based initially on pharmacological criteria (3–5). Each class has separate, but overlapping physiological roles and corresponding links to various pathologies in the nervous system. Dysfunction among all three classes has been implicated in epilepsy (6), Parkinson’s disease and Alzheimer’s disease, (7), as well as excitotoxic neuronal death (8). Additionally, NMDA receptors are thought to be involved in chronic pain (9) and mechanisms of drug and alcohol addiction (10, 11). NMDA receptors have many properties that distinguish them from other members of the glutamate receptor superfamily (i.e., voltage-dependent Mg2+ inhibition, permeability to Ca2+, and the requirement of the co-agonists glycine or D-serine), and multiple NMDA receptor subunit genes contribute to their functional diversity (12). The NR1 subunit, which binds glycine, has eight splice variants, and is ubiquitously expressed in the central nervous system (5). Four separate genes (NR2A-NR2D) encode different isoforms of the NR2 subunit, which binds glutamate; expression of different NR2 genes varies according to both regional and temporal expression patterns in the brain (12). In addition, two NR3 genes have been identified (NR3A-NR3B), which also vary in their regional and temporal expression patterns (13). The assembly of a functional tetrameric complex is thought to require the presence of two obligatory NR1 subunits and a combination of modulatory NR2 or NR3 subunits; studies examining receptor subunit assembly indicate that NR1 subunits form heterodimers with NR2 or NR3 subunits at the initial stage in assembly (14). Although much remains to be elucidated, pharmacological evidence suggests that different NMDA receptor subunit combinations may play different roles in the physiology and corresponding pathology of the central nervous system. For instance, NMDA receptor antagonists that selectively inhibit receptors containing the NR2B subunit have been implicated in the treatment of epilepsy and chronic pain, whereas more broadly selective NMDA receptor antagonists generally result in toxicity or intolerable side effects in the same assays (15). Conversely, more broadly targeted NMDA receptor antagonists were effective at preventing axonal de-myelination in models of ischemia, whereas NR2B-specific antagonists had no effect on myelin degradation, suggesting the antagonism of non-NR2B subtypes mediates this protective effect (16). Interestingly, evidence suggests the effects of NR2B-selective antagonists show correspondence to temporal and regional expression patterns in the brain. For instance, NR2B expression is enriched in hippocampal and temporal regions in adult rats (17), corresponding to areas of the brain affected in many types of epilepsy, and suggesting a possible mechanism of action. It remains to be determined whether antagonists targeted to other NR2 subunits, such as NR2D, which is enriched in midbrain regions (12), affect the physiology and pathophysiology associated with these brain regions. In order to understand the function of each individual receptor subtype in the intact nervous system, the standard approach that has been employed is gene knockout technology. As is the case for all ion channel complexes with multiple subunits, glutamate receptor gene knockouts need to be complemented with pharmacological agents that are as highly subtype selective as possible. It is particularly desirable to find ligands which bind at subunit interface sites that permit the ligand to interact with determinants on two different subunits. There is a fundamental difficulty with using knockout technology exclusively to assess ion channel function for those families in which a wide variety of different receptor subunits can be assembled as heteromeric complexes in vivo. Ablating the function of a gene that encodes a single type of subunit can result in a very complex phenotype, in which multiple functional complexes with different subunit compositions and physiological roles are affected. The phenotype of NMDA receptor NR1 subunit gene knockout mice, for instance, is not straightforward – concomitant expression of the NR2B subunit is markedly reduced, and neonates do not survive long after birth (18). One often-effective approach to obtaining subtype selective pharmacological agents to complement gene knockout technology is to start with the pharmacologically-active components of animal venoms. Unfortunately, there have not been a large number of venom components characterized to date that target glutamate receptors. The polyamine toxins found in spider venoms are one such class (19). A promising general source that needs to be explored further are the venoms of cone snails; these have proven to be very rich in families of peptides targeted to ion channels. There are two Conus peptide families that target glutamate receptors identified so far, the conantokins, that act as NMDA receptor antagonists (15, 20–22), and the recently identified con-ikot-ikots, which are targeted to AMPA receptors (C. Walker et. al., submitted for publication). All members of the conantokin family target NMDA receptors, but with differing subtype selectivity; conversely, the con-ikot-ikots appear to exclusively target AMPA receptors, although many properties of this peptide family remain to be investigated (23–25). For both these families, the number of Conus species that have been identified with venom components targeted to glutamate receptors has been surprisingly small. Among the conantokins, peptides from only six species have been characterized (20, 24–28), and within the con-ikot-ikot family, a peptide from a single Conus species has been characterized. We recently initiated an expanded effort to systematically examine new Conus species (29) for venom components that selectively target glutamate receptor subtypes. Previous work has used synthetic peptide analogues to identify some of the determinants of NR2A and NR2B subunit selectivity in conantokins (30) (reviewed in 31). To expand upon this approach we have begun to search for sequence variations among native conantokins that govern target specificity. In this report, we describe a new peptide, conantokin-Br, from Conus brettinghami (1) that has a number of properties that are novel, including higher relative potency for the NR2D subunit than has been previously reported for conantokin peptides. We also characterized chimeras between conantokin-Br and a conantokin with different subunit selectivity, conantokin-R. This provided an opportunity to assess the relative importance of primary sequence variations with respect to activity on different NMDA receptor subtypes. This study has led to a number of structure/function insights for developing ligands with divergent selectivity for different NMDA receptor subtypes based on the conantokin family of conopeptides.