IDENTIFICATION OF KEY FUNCTIONAL DOMAINS IN CYS-LOOP RECEPTORS

GUMILAR FERNANDA; RAYES DIEGO; SINE STEVEN; BOUZAT CECILIA

Bariloche, Río Negro, Argentina.

Congreso; XXXIX Reunión Nacional de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2003

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB),Sociedad Argentina de Biofísica (SAB)

The AChR, 5-HT3 and GABAA receptors are members of the  cys-loop superfamily of ligand-gated ion channels that  mediate rapid synaptic transmission throughout the nervous system. Neurotransmitters interact with a ligand-binding site in these channels triggering a conformational change in the protein that results in the opening of an ion channel. The structure of an homopentameric soluble protein (AChBP) from Lymnaea stagnalis, which is secreted by snail glial cells into cholinergic synapses,  has been recently described at a high resolution. AChBP binds agonists and competitive antagonists of the AChR and its sequence is 20-24% identical to aligned sequences of the amino-terminal, extracellular halves of AChR subunits. To ascertain whether AChBP exhibits the potential to function as the extracellular region of a LGIC, and to identify extracellular domains involved in coupling the recognition of agonist to the opening of the channel, we constructed a chimeric AChBP-5HT3 subunit and expressed it in mammalian cells. The chimeric receptor shows high expression and the same pharmacological profile as that of the soluble AChBP, indicating that the overall structure of the binding domain is conserved. However, results from macroscopic current recordings show that channel opening is not efficient in this receptor. Exchanging the cys-loop of AChBP by that of 5-HT3 leads to the lack of expression, suggesting that this domain is essential for appropriate protein folding. The replacement of domains in AChBP until gating is achieved is our current strategy to identify residues involved in channel activation.