CAREER: Conformational Changes in Voltage-Controlled Ion Channels Measured by Advanced Fluorescence Techniques

University Of Illinois At Urbana-Champaign, Urbana IL

Investigators

Abstract

This project describes an interdisciplinary five-year career development plan involving physics, chemistry, and biology in both research and teaching. In research, two complementary fluorescence techniques will be used to study conformational changes in ion channels-channels that are responsible for nerve conduction. These techniques, one of which measures distance and the other of which measures rotation, can be applied to many other kinds of proteins and channels. The first technique, lanthanide-based resonance energy transfer (LRET), is an improved version of an established technique called fluorescence resonance energy transfer. This improved resolution is achieved because the lanthanide can transfer energy to an organic dye by a mechanism that is sensitive to angstrom changes in distance between the two labels. The second method is a version of phosphorescence anisotropy utilizing either phosphorescent probes or our recently discovered polarized luminescent lanthanide probes. In this technique, a polarized excitation pulse selectively excites those probes aligned with the pulse polarization and polarization-sensitive detection monitors the reorientation of probes. The technique has the advantage that it can measure rotational motion on the millisecond time scale, the time-scale associated with protein rearrangements in ion channels. LRET will be used to measure distances between site-specifically placed lanthanide atoms and organic dyes as a function of voltage across the cell membrane and relate them to motion within the channel during simultaneous electrical measurements. Measuring rotation of appropriately placed probes by phosphorescence anisotropy can further characterize protein movement. Initial studies with LRET have achieved a milestone in the field of voltage-controlled ion channels, the first measurements of actual distance changes as ion channel proteins move in response to voltage changes. These results suggest that part of the channel rotates, a hypothesis that will be tested further. In teaching, an undergraduate course intended primarily for non-scientists, entitled "Science and the Citizen: Current controversies in science, technology and society" will be developed. The course will be taught in a "pro-con" format with introductory lectures giving students background in contemporary science issues, followed by readings from advocates of both sides and extensive student-centered debate and writing to enhance students' critical, analytical, and communication skills. Topics will include: the feasibility and desirability of a ballistic missile defense system, environmental issues such as the ozone hole and global warming, health issues such as pesticides in food, chemicals in the environment, genetic engineering, and cloning animals.