The GABAA receptors are members of the neurotransmitter-gated ion channel gene superfamily that includes nicotinic acetylcholine, serotonin 5-HT3 and glycine receptors. The GABAA receptors mediate fast inhibitory synaptic transmission in the central nervous system. A long term goal of molecular neuroscience has been to understand the structural bases for the functional properties of these receptors and for their modulation by clinically used medicines and by drugs of abuse. Structure-function studies of these receptors took a quantum leap on June 25, 2003 with the publication of a 4Angstrom resolution closed state structure of the homologous 'Torpedo' acetylcholine receptor (AChR) (Miyazawa et al., 2003). At 4A resolution this structure provides a solid foundation for future studies of protein dynamics and agonist-induced conformational changes. At 4Angstrom resolution the peptide backbone path is well defined but the individual amino acid side chain positions are poorly defined. As expected, in the transmembrane (TM) domain each subunit contains four alpha-helical segments (M1, M2, M3, M4) with the M2 segment forming the channel lining and gate. Surprisingly, the helical TM segments extend approximately 10Angstrom above the extracellular membrane surface where they interact with the largely beta-strand, extracellular, agonist-binding domain. A critical interaction between the extracellular and TM domains is via a residue in extracellular Loop 2 and residues in the M2-M3 loop. This proposal will focus on three aspects of GABAA receptor structure: 1) verifying the applicability of the AChR structure to the GABAA receptor, 2) studying the dynamic motion of the channel in the closed state and 3) studying the conformational changes that occur during channel gating from the closed to the open/desensitized states.
Aim #1 will test the hypothesis that the AChR structure is a good model for the GABAA receptor by testing predicted proximity relationships between the M2 and M3 and between the M2 and M1 segments within a subunit.
Aim #2 will test Unwin and colleagues' gating hypothesis.
Aim #3 will probe changes in the M2 segment tertiary and quaternary structure during gating.
Aim #4 will probe the tightness of protein packing around the extracellular half of the M2 segment from the 12' to the 27' levels.
Aim #5 will probe the mobility and flexibility of the extracellular helical extension of the M2 segment from the 21' to the 27' level. Successful completion of the proposed experiments will either confirm the AChR structure or will provide an experimental basis for refining the structure. In addition, completion of the proposed experiments will provide new insights into the dynamics of the GABAA receptor channel-lining domain in the resting state and as the channel undergoes its agonist-induced conformational changes.
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