Deciphering the function of neurotransmitter-gated ion channels: From the molecular to the animal level

BOUZAT, C.

Capital Federal, Argentina

Simposio; Segundo Simposio Franco-Argentino en Neurociencias; 2012

Rapid communication in the nervous system is mediated by neurotransmitter-gated ion channels. Cys-loop receptors are members of this superfamily that includes serotonin (5-HT3) and acetylcholine receptors (AChR). They are pentameric proteins composed of a large extracellular domain that carries the binding sites and a transmembrane region that contains the ion pore. Communication over the 50 Å separating the binding site and the pore is thus central to activating Cys-loop receptors. To delineate molecular mechanisms and structures underlying function we apply molecular biological methods to manipulate receptor structure and expression in mammalian cells, high resolution single-channel and macroscopic current recordings to determine kinetics of activation and desensitization and mechanisms of drug modulation, and in silico studies. We focus on homomeric receptors, such as a7 AChR and 5-HT3A, because as they are the simplest structural members of the family they are invaluable models for structure-function studies. To determine how many of the five agonist binding sites are required to be occupied by the agonist for receptor activation, we used the homomeric receptor model, a7-5HT3A. We have developed a strategy, called electrical fingerprinting, that allows determination of the number of subunits in the pentameric receptor required for a given functional process. By applying this strategy, we found that agonist occupancy of only one of the five functional binding sites of a homomeric receptor allows channel opening although the open channel is unstable; and occupancy of three non-consecutive sites is required for maximal open channel lifetime. During activation, the initial conformational changes at the binding site are propagated to the pore. By analyzing activation and desensitization kinetics of a series of a7/5-HT3 chimeric receptors, we demonstrated that the interface between extracellular and transmembrane domains, called coupling region, forms a network of interdependent loops that allows functional connection between the binding site and the gate, and it also contributes to channel lifetime and desensitization. Thus, this region governs the beginning, duration and refractory period of synaptic responses mediated by Cys-loop receptors. C. elegans is an excellent model for the study of synaptic transmission and it is also a model of nematode parasites. Muscle L-AChRs are of clinical interest since they are targets of anthelmintic drugs. By measuring currents in cultured muscle cells from a series of C. elegans strains lacking AChR subunits, we determined the subunit composition of L-AChRs. Our complementary in silico studies provide testable hypotheses for elucidating the subunit arrangement and identifying determinants of anthelmintic selectivity. Generation and analysis of strains carrying mutations in L-AChR subunits which mimic those found in patients suffering from congenital myasthenic syndromes (CMS) have revealed that C. elegans is a valid model for human CMS and for drug testing.