| Glutamate receptors (GluRs) are ligand gated ion channels. They bind glutamate, the major excitatory neurotransmitter in the brain, and are essential to normal brain function and, when dysfunctional, contribute to numerous brain diseases. GluRs are integral membrane proteins with four subunits per functional receptor. Each subunit has four domains each with independent evolutionary origin: the amino terminal and ligand binding domain are extracellular, the transmembrane domain is located in the lipid bilayer, and C-terminal domain is intracellular. The transmembrane domain includes 4 hydrophobic segments, M1-M4, with M1 to M3 forming the ion channel core and having a structural homology to two transmembrane K+ channels but with an inverted orientation in the membrane. Supporting this relationship are two transmembrane prokaryotic GluRs (e.g., GluR0) that are functional though they display gating kinetics quite different from mammalian GluRs. The M4 segment (as well as the C-terminal domain) has an unknown evolutionary origin. During my thesis I studied the structural and functional significance of the additional transmembrane segment, M4, in mammalian GluRs.;Mammalian GluRs need the additional transmembrane segment (M4) to function, in contrast to prokaryotic GluRs. Specifically, I found that deletion of M4 in AMPA (GluR-A) receptors abolishes glutamate-activated current although the receptor expresses at the membrane. This lack of functionality is not due to any apparent interaction of M4 with the ligand-binding domain since decoupling M4 from the ligand-binding domain by introducing multiple glycines into the linker (joining M4 to the ligand-binding domain) has no notable effect on function. In contrast, mutagenesis scans of M4 as well as recovery of function from polyleucine M4 transmembrane helped us to define a unique face of the M4 helix that is required for glutamate receptor function. These interactions are in part involved in gating transitions in transmembrane segments. Hence, my work indicates that the interaction of M4 with other transmembrane segments is required for channel gating in mammalian GluRs, presumably to allow key gating elements (M3 and/or M1) to undergo their conformational change. |
