We wish to understand at the systems level the role of specific ion channels in electrogenesis and nerve cell conduction. Some of the underlying questions that we are attempting to answer include: 1) what is the rate at which channels are being made and degraded, and are such rates context dependent (i.e., is protein turnover affected by increased neural activity, nerve cell damage, etc.); 2) how are the membrane concentrations and cellular distributions of functional channels changing as a result of nerve insult; and 3) is channel function regulated by neighboring glial cells. Answers to these types of questions will better inform our understanding of molecular mechanisms by which nerve cells respond to external stimuli and to injury.
While small molecule chemical design and synthesis play an integral role in this evolving story, as a lab, we are resolved to use any tool necessary for our investigations. We conduct experiments across all facets of this work, which include complex small molecule design and synthesis, molecular and cellular biology, electrophysiology, fluorescent imaging, and neurobiology. Our investigations are uniquely enabled through collaborations with the Cui, Huguenard (Neurology & Neurosurgery), Maduke (Molecular and Cellular Physiology), Shah (Psychiatry & Neurobiology), and Zuchero (Neurosurgery) labs.
Chloride channels play critical roles in the electrical excitation of muscles and neurons, as well as in maintaining the proper water and salt balance in the body. Our lab has developed several small molecules with unprecedented potency and selectivity for single chloride channel isoforms. This will allow us to look into the roles of specific chloride channel isoforms.
Voltage-gated sodium channels (NaVs) are responsible for the rising phase of the action potential. Their dysregulation is implicated in multiple channelopathies, including heart arrhythmia, epilepsy, and neuropathic pain. Our lab has developed total syntheses for several potent sodium channel agonists and inhibitors, including the neurotoxins saxitoxin, tetrodotoxin, and batrachotoxin. We are currently derivatizing these toxins to study channel biophysics, label membrane-bound NaVs, and selectively modulate channel function. Selected examples of work in each area are shown below.
Synthetic batrachotoxin-B (BTX-B) and its enantiomer share the same binding site. However, they exert markedly different effects on channel function, with BTX-B acting as a channel agonist, and ent-BTX-B as an antagonist. We are using these toxins to better understand channel gating and dynamics, as well as to investigate how small molecule toxins can exert drastic effects on these channel properties.
Modification of saxitoxin with a reactive maleimide functional group has allowed us to covalently modify membrane-bound sodium channels. We are currently developing these probes into imaging agents, for the purpose of tracking NaV expression, trafficking, and turnover in live cells.
Mutant cycle analysis
By comparing the affinities of several saxitoxin derivatives with various sodium channel mutants, we have identified key contacts between saxitoxin and its binding site. Modified toxin-mutant channel pairs can be used to functionally ‘knock out’ specific channel subtypes, allowing for investigations into the function of individual isoforms.