Volatile anesthetics (VAs) are a clinically important class of drugs whose mechanism of action is not understood. Genetics is an essential tool for determining which molecular events actually produce anesthesia. Genetic screens in the model organism Caenorhabditis elegans identified an unusual mutation in the syntaxin-1 A gene rendering animals VA-resistant. Given the highly conserved nature of the presynaptic proteins through which syntaxin-1 A functions, this presynaptic VA mechanism is likely to be operant in humans. The broad goals of this proposal are to identify the conserved VA targets and to understand how VAs alter their function.
Our specific aims are: 1) Define genes essential for presvnaptic volatile anesthetic action in C. eleoans. We will use genetic methods to test the role in VA mechanisms of specific proteins that function with syntaxin. In particular, the highly conserved UNC-13 protein is a strong candidate as an anesthetic target. 2) Identify additional components of the volatile anesthetic mechanism by genetic screens. We have recently screened for new mutants that are VA resistant. We propose to identify the mutated genes. This approach has the potential to discover novel VA targets and clarify the presynaptic mechanism studied in aims 1 and 3. We also will test the role of two-pore potassium channels in C. elegans by screening through knockouts or RNAi-knockdowns of TASK-1, TASK-3, and TREK-1 homologs. Identifying C. elegans K* channel VA targets will aid in defining domains necessary for VA action on these channels in vertebrates. 3) Define the role of candidate proteins in the presvnaptic VA mechanism by combining electrophvsioloqy with genetics. In collaboration with Janet Richmond, the leading expert in C. elegans electrophysiology, we have shown by electrophysiological methods that VAs inhibit transmitter release in C. elegans. We will use these electrophysiological techniques to define the mechanisms whereby the identified genes control VA sensitivity. In addition because of constraints of behavioral assays, electrophysiology will provide the only means to test the role of some proteins in the VA mechanism. The combination of genetics and electrophysiology is synergistic and essential to define the relevant VA targets and how VAs affect their function. ? ?
