Identifying Genes Required for the Regulation and Function of Ionotropic Glutamate Receptors.

In order to form a functional nervous system, embryonic neurons must differentiate into a diverse number of distinct neuronal types defined by morphology, connectivity and the specific complement of expressed molecules.  For example, excitatory ionotropic glutamate receptors (iGluRs) must be expressed in the correct neurons and localized to the appropriate synapses – specialized sites of contact between neurons.  Our goal is to identify key molecules required for these processes.

Functional iGluRs are ligand-gated ion channels formed by a heteromeric assembly of 4 glutamate receptor subunits.  Two major families of iGluRs have been described based on pharmacological specificity – the NMDA receptors and the non-NMDA receptors, the latter including both AMPA and kainate subtypes.  In vertebrates, 4 AMPA subunits (GluR1-4), 5 kainate subunits (GluR5-7, KA1 and KA2), and 7 NMDA subunits (NR1, NR2A-D, NR3A and NR3B) have been described (Dingledine, 1999).  An orphan family of subunits, GluRd1 and GluRd2, has been identified, but not well characterized.  C. elegans expresses at least 10 iGluR subunits that have been assigned to either the non-NMDA family (GLR-1 – GLR-8) or the NMDA family (NMR-1 and NMR-2) (Brockie and Maricq, 2003).  To understand how these subunits are assembled into functional receptors and localized to the correct synapses, we have designed genetic screens to isolate mutants with defects in behaviors dependent on iGluRs.

We generated a transgenic strain that expresses a constitutively active or ”leaky” variant of the GLR-1 subunit (Zheng et al., 2004).  These worms are referred to as “lurcher” worms as the leaky GLR-1 subunit has an alanine to threonine mutation analogous to that identified in the GluRd2 subunit in the lurcher mutant mouse (Zuo et al., 1997).  The GLR-1 lurcher channel causes dramatic defects in locomotion where transgenic worms rapidly switch between forward and backward locomotion.  By screening for mutations that suppress this hyper-reversal phenotype, we hope to identify molecules required for receptor expression, assembly, trafficking/targeting, membrane insertion, and ligand-gating.  We have described the first gene identified in the suppressor screen – sol-1.  Although sol-1 is not necessary for receptor expression, trafficking or membrane insertion, electrophysiological analysis of glutamate-gated currents in C. elegans interneurons in vivo suggests that SOL-1 is required for receptor gating (Zheng et al., 2004).

We would also like to identify genes required for NMDA receptor function using a similar strategy to express constitutively active variants of the NMDA receptor subunits NMR-1 and NMR-2. The screens are predicted to identify genes required for the proper function of iGluRs and thus further our understanding of the mechanisms that form a functional glutamatergic synapse.

Brockie, P.J., and Maricq, A.V. (2003).  Ionotropic Glutamate Receptors in Caenorhabditis elegans.  NeuroSignals. 12(3), 108-25.

Dingledine, R., Borges, K., Bowie, D., and Traynelis, S.F. (1999).  The glutamate receptor ion channels.  Pharmacol Rev. 51(1), 7-61.

Zheng, Y., Mellem, J.E., Brockie, P.J., Madsen, D.M., and Maricq, A.V. (2004)  SOL-1 is a CUB-Domain Protein Required for GLR-1 Glutamate Receptor Function in C. elegans. Nature 427, 451-457.

Zuo, J., De Jager, P.L., Takahashi, K.A., Jiang, W., Linden, D.J., and Heintz, N. (1997). Neurodegeneration in Lurcher mice caused by mutation in delta2 glutamate receptor gene. Nature 388, 769-73.

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