The Cys-loop receptor family includes the nicotinic acetylcholine receptor (nAChR) and the serotonin type 3 receptor (5-HT3R). Cys-loop receptors are neurotransmitter-activated ion channels participating in neurotransmission in the nervous system. They are targets of drugs for treating disorders from nausea to anxiety. Mutations in Cys-loop receptors can lead to diseases including epilepsy. Neurotransmitter binding opens the ion channel by initiating the movement of the second transmembrane (TM2) domain of each of the channel's five subunits. In the conventional view of the channel an outer vestibule narrows to the rate-limiting region within the membrane where the gate is located; the pore then opens into the cytoplasm. Thus, in this scheme, determinants of ion conduction reside exclusively in TM2. However, recent evidence renders this textbook view of Cys-loop receptors obsolete: First, cryoelectron microscopy revealed that the nAChR has narrow "portals" at the cytoplasmic interfaces of each subunit; Second, mutagenesis of these regions in the 5-HT3R influences ion conduction. These findings necessitate revision of the model for the ion permeation pathway. Thus this project addresses a fundamental question in neuroscience: does conduction through ion channels involve cytoplasmic elements? Data from this laboratory demonstrate that intracellular regions of 5-HT¬3Rs affect all aspects of ion conduction: conductance, voltage-dependence and ionic selectivity. This study will identify the mechanisms involved, creating a new model for Cys-loop receptor function. A key objective of the study is to continue to develop a Cys-loop receptor database accessible to physicians, researchers and educators. Thus this project will enhance the infrastructure for research and education at The George Washington University and beyond. Undergraduate students spearhead the development of the Cys-loop receptor database.
Cys-loop receptors are neurotransmitter (e.g. serotonin and acetylcholine) activated ion channels that mediate neurotransmission in the vertebrate central and peripheral nervous systems. Mutations that adversely affect ion conduction through these channels can have important neurological consequences. Attempts to determine the complete structure of Cys-loop receptors have so far been unsuccessful because most of their intracellular portion is intractable to structural analysis. We have demonstrated that this part of the protein is very important from a functional perspective influencing the rate, selectivity and voltage-dependence of ion conduction as well as protein trafficking and neurotransmitter efficacy. The most complete structural model comes from cryo-electron microscopic analysis of the Torpedo marmorata nicotinic acetylcholine (nACh) receptor. We used this template to target mutagenesis, systematically probing the functional contribution of intracellular amino acids using electrophysiological techniques to record neurotransmitter activated microscopic and macroscopic currents. The textbook view of Cys-loop receptors depicts these pentameric proteins with a large extracellular portion coupled to a cylindrical pore formed by adjacent second membrane-spanning domains (TM2) of each subunit, which contain all the determinants of ion conduction. However, several studies, including our work on serotonin-activated receptors and nACh receptors, have demonstrated that this depiction is too simplistic. Narrow structures (portals) at the cytoplasmic interfaces of subunits form part of the conduction pathway. Mutagenesis within the so called membrane-associated (MA)-stretch in each subunit’s cytoplasmic TM3-4 loop influences the rate of ion conduction. Our studies of serotonin-activated receptors reveal that the charge of specific amino acids within the MA-stretch determines both the rate of ion conduction and the ionic selectivity, while amino acid volume influences rectification. The same residue is a determinant of the rate of ion conduction through nACh receptors. Our structural models of these mammalian receptors based on the T. marmorata nACh receptor structure reveal the location of MA-stretch residues at the entrances of cytoplasmic portals. Our work continues to refine the structural model of Cys-loop receptors emphasizing the importance of the intracellular portion of these proteins. To make our findings and those of other investigators more accessible to educators and researchers, the results of mutagenesis studies are being compiled in a web database (www.cys-loop.org) that is becoming an important research and educational resource.
