STRUCTURAL AND FUNCTIONAL ANALYSES OF A NOVEL INTERACTION BETWEEN GABA-A RECEPTORS AND CAML

Open Access
- Author:
- Yuan, Xu
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 19, 2007
- Committee Members:
- Richard W Ordway, Committee Chair/Co-Chair
Bernhard Luscher, Committee Member
Gong Chen, Committee Member
Douglas Cavener, Committee Member
Pamela Hankey Giblin, Committee Member - Keywords:
- calcium modulating cyclophilin ligand
trafficking
GABA-A receptor - Abstract:
- ABSTRACT Inhibitory synaptic transmission in the brain is mainly mediated by heteropentameric GABAA receptors (GABAARs) composed of ƒÑ and ƒÒ subunits together with the ƒ×2 subunit. The postsynaptic clustering of these receptors is critical for the efficacy of synaptic transmission. Moreover, trafficking and targeting of GABAARs to the postsynaptic membrane is highly regulated, and this mechanism is thought to contribute to functional plasticity of GABAergic synapses. The ƒ×2 subunit contributes to the majority of GABAAR subtypes and is essential for clustering of GABAARs at postsynaptic sites, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic synapses. Targeted deletion of the ƒ×2 subunit gene leads to loss of major postsynaptic GABAAR subtypes in vivo (Essrich et al., 1998; Schweizer et al., 2003). And the fourth transmembrane region (TM4) of the ƒ×2 subunit is thought to play an important role in this mechanism (Alldred et al., 2005). To elucidate the mechanism by which the ƒ×2 subunit contributes to trafficking of GABAARs, a yeast two hybrid screen was performed to identify novel proteins that interact with the TM4 and cytoplasmic region of the ƒ×2 subunit. As part of my Ph. D. thesis I here report the isolation of calcium-modulating cyclophilin ligand (CAML) as a novel GABAA receptor-interacting protein. CAML is an integral membrane protein first identified as endogenous ligand for cyclophilin B, the receptor of the immunosuppressant cyclosporin A (Bram and Crabtree, 1994) and subsequently shown to be essential for the endocytic recycling of internalized epidermal growth factor receptors (EGFRs) to the plasma membrane (Tran et al., 2003). Using yeast two-hybrid tests the ƒ×2 subunit-interaction domain has been mapped to the N-terminal cytoplasmic domain of CAML. Conversely, both the 3¡¦ half of the major cytoplasmic loop domain and the TM4 domain of the ƒ×2 subunit were required for interaction with CAML. This novel interaction between CAML and the ƒ×2 subunit was verified by colocalization assays and immunoprecipitations in heterologous cells, by colocalization of transfected and endogenous proteins in neurons and by co-immunoprecipitation of native proteins from brain extracts. The role of CAML in trafficking and clustering of postsynaptic GABAAR subtypes was analyzed by disrupting the function of CAML through RNA interference (RNAi) in cultured neurons and by conditional knock-out of a floxed CAML gene in cultured neurons and mice. Following knockdown of CAML by short hairpin RNA (shRNA) based RNAi, postsynaptic clusters of GABAARs were decreased concurrent with a similar reduction in immunoreactivity representing GABAergic presynaptic terminals. Inactivation of the CAML gene in mouse embryos (Emx1Cre x CAMLf/-) resulted in neonatal lethality and neurons cultured from corresponding embryos died in vitro. Postnatal deletion of CAML in CaMKIICre2834 x CAMLf/- mice resulted in loss of ƒ×2 subunit-containing GABAARs as indicated by a reduction in 3[H]-flumazenil binding in brain sections analyzed by autoradiography. Tamoxifen-induced inactivation of CAML in neurons cultured from CAGGCre-ERTM x CAMLf/- embryos lead to a small but significant reduction in the number and size of ƒ×2 subunit clusters. Biotinylation assays indicated normal endocytosis of GABAARs from the cell surface with, however, a significant deficit in recycling of endocytosed receptors to the cell surface. Eletrophysiological analyses of CAML-deficient neurons (tamoxifen treated CAGGCre-ERTM x CAMLf/- neurons and CAML shRNA-transfected neurons, compared to respective controls, revealed a significant reduction in GABA-induced whole cell current densities as well as reduced frequency in spontaneous and miniature inhibitory postsynaptic currents. All together, our data suggest that CAML is critical for neural differentiation, for postsynaptic clustering, and for normal endocytic recycling of ƒ×2 subunit containing GABAARs.