Cys-loop Receptors: from structure to function

BOUZAT C

Rosario

Conferencia; Reunion Anual Sociedad Argentina de Biofisica; 2006

Sociedad Argentina de Biofisica (SAB)

Cys-loop receptors, such as nicotinic (AChR), 5HT3, GABA and glycine receptors, mediate rapid synaptic transmission throughout the nervous system. Their essential function is to couple the binding of agonist at the extracellular domain to the opening of an intrinsic ion pore. This mechanism, which is known as gating, has not been yet elucidated. To investigate the structural basis for functionally coupling binding and pore domains, we generated a chimeric receptor composed of the acetylcholine binding protein (AChBP) and the pore domain from the 5-HT3A receptor. The chimeric AChBP-5HT3A receptor shows high surface expression on mammalian cells but it does not function, suggesting that although binding and pore domains are corrected folded, the interface between them is not compatible, preventing inter-domain coupling. Only when amino-acid sequences of three loops in AChBP (b1b2, cys and b8b9) are changed to their 5HT3A counterparts does ACh bind with low affinity characteristic of the activatable receptor and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modeling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process. Each subunit of all Cys-loop receptors is composed by an extracellular domain that contains the binding sites and four transmembrane (TM) segments (M1 ¨C M4). The M2 segment of each subunit delineates the ion channel pore, which contains the gate that allows the pore to switch from ion-impermeable to ion-permeable. The role of the other TM segments in channel gating is less understood. By combining site-directed mutagenesis with single-channel kinetic analysis we explored the contribution of M1, M3 and M4 domains to channel gating. Our results reveal that these segments affect opening and closing rates and that the structural bases of their contributions to gating are specific to each subunit.  The atomic model of the closed pore of the AChR shows that although M2 makes no extensive van der Waals contacts with the other transmembrane segments, there are several sites where close appositions between segments occur. It has been suggested that the pair aM1-F15¡ä and aM2-L11¡ä is one of the potential interactions between segments. To determine experimentally if these residues are interacting and to explore if this interhelical interaction is essential for channel gating, we combined mutagenesis with single-channel recordings. Replacing the residue at aM1-15¡ä by that at aM2-11¡ä and vice versa profoundly alters gating, but the combination of the two mutations restores gating to near normal, indicating that aM1-F15¡ä and aM2-L11¡ä are interchangeable. Double-mutant cycle analysis shows that these residues are energetically coupled. Thus, this interaction is essential for appropriate channel gating probably by connecting the M2 movement to the M1 movement. Finally, understanding how the transmembrane helices interact with each other will help us to understand how the assembled receptors carry out their biological functions.