Research Expertise and Interest
Ion channel function, synaptic plasticity, neural excitability, synaptic transmission, the synapse
The work in the lab is focused on ion channels and other signaling proteins, and their role in neural excitability, synaptic transmission, and the molecular and functional development of the synapse.
Structure and protein motions of voltage and ligand gated channels. We have developed new methods for studying ion channel structure and protein motion. We label channel proteins on the surface of living cells in a site-directed manner with fluorescent dyes. We detect local protein motion via changes in the microenvironment of the dye in real-time, with sub-millisecond resolution, by measuring fluorescence intensity, wavelength and dye mobility. These optical reports identify "what moves when" in relation to functional events that are synchronized for the channel population and measured with a voltage clamp. In addition to obtaining kinetic maps of protein motion, we use fluorescence resonance energy transfer as a spectroscopic ruler to make structural determinations. This method enables us to view movements of individual segments of the channel protein, in individual subunits, so that the spatial location and role in gating of each domain of the protein can be determined. From measurements of movement and distance we are working to assemble an animated structural model of the conformational rearrangements that activate, open, close and inactivate channels. In addition, we are now beginning to make optical measurements of protein motion on the single channel level. Our work, which began with a voltage-gated K+ channel, is now expanding to other classes of channels that are activated by very different classes of signals, such as ligand binding. Our aim is to discover general principles that enable different kinds of signal transduction apparatus to control the conformation of molecular gates that control ion flux and ion selectivity.
Development of genetically encoded optical sensors of cell signaling. Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural signaling. To address this problem, we have constructed a novel, genetically-encoded probe that can be used to measure transmembrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into the voltage-sensitive Shaker channel, creating a channel whose voltage-dependent rearrangements induce large changes in the fluorescence of GFP. A genetically manipulable voltage sensor has the advantage that its operating range can be tuned by mutagenesis, and that it may be introduced into an organism and targeted for expression to specific developmental stages, brain regions, cell types, and sub-cellular locations. We are using similar strategies to develop different optical sensors by fusing this GFP to other detector proteins such as channels and receptors. Such sensors can be used to ask specific biological questions about the native role of the detector proteins, or, instead, to report on the signals that activate these proteins. We are using such sensors to determine the role of signaling pathways in Drosophila synapse formation and in several other model synaptic systems.