Electrified cryo-EM: a new tool to capture metastable neuron structures during an action potential

Project Summary:

There remains a critical blind spot in the study of biological systems. In situ imaging techniques like super-resolution microscopy can elucidate cellular dynamics but cannot provide the sub-nanometer resolution needed for molecular detail. Cryogenic electron microscopy (cryo-EM) does provide atomic-resolution structures yet remains limited to

only systems in their equilibrium states. While both these techniques have been awarded separate Nobel prizes for their transformative impact on our understanding of biology, a missing gap still exists for how the structure of biomolecules change in their active state far from equilibrium. The proposed research seeks to bridge this gap. This

project will pioneer the development of electrified cryo-EM (eCryo-EM), a novel tool developed by my research

group that can kinetically trap biological systems in their metastable state away from equilibrium. Briefly, an electrical stimulus applied just prior to and throughout a plunge-freezing step will capture and preserve metastable states in electrically excitable biological systems. This approach in using eCryo-EM to capture metastable state is general for

all of biology. Indeed, many fundamental questions remain unanswered for a broad spectrum of electrically excitable biological systems with important implications for medical applications (e.g., wound healing, electroporation drug

delivery, muscle contraction, sensory acquisition, etc.). As a starting point, this proposal will unravel the first snapshots of neuron conformational changes through the stages of an action potential across multiple length scales. At the molecular level, eCryo-EM will reveal the molecular structure of voltage-gated channel proteins as they change

through open, closed, and inactivated states. At the cell level, eCryo-EM will reveal the mechanism of neural inhibition at the synaptic cleft under high-frequency waveforms. These new insights not only provide fundamental understanding for neuron behavior under an electrical stimulus, but also will guide future neurostimulation treatments

that are directed and tailored for a specific neural circuit to restore human health in patients suffering from neurological disease. 3 tasks are outlined to achieve the stated goals: (1) demonstration of an eCryo-EM device that

can successfully trap metastable states of biological systems under an electrical stimulus; (2) elucidating a time series of structural changes in voltage-gated channel proteins throughout an action potential; and (3) trapping the multiple conformations of a synaptic junction between neurons throughout electrical stimulation. These tasks cannot be

achieved through conventional methodologies. Rather, it requires a fundamentally new approach in kinetically

trapping the active state biological systems directly under an electrical bias. The proposed research will establish a new paradigm in investigating how neurons fundamentally behave in response to an external electrical stimulus, thereby providing new neurostimulation therapeutic targets and strategies for treating neurological diseases.