Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Wednesday Speaker Abstracts

Combining Electrical and Optical Measurements on Voltage-gated Potassium Channels Corianne Van den Akker , Steven Boxer. Stanford University, Stanford, CA, USA. The structure, precise operation mechanism and dynamics of ion channels have not yet been elucidated, because linking the structure and structure changes to function has remained challenging. We are developing new methods that allow the structure of membrane proteins to be studied in a controlled, native-like environment by simultaneously probing their structure and function. We report a second-generation membrane interferometer (Ganesan et al., Proc. Natl. Acad. Sci. 106, 2008) in which electrophysiology measurements can be performed simultaneously with high-resolution fluorescence microscopy imaging. In the membrane interferometer, a freestanding bilayer is formed over a micropore that is positioned above a reflective mirror. The mirror allows the use of Fluorescence Interference Contrast (FLIC) and Variable Incidence Angle-FLIC (VIA-FLIC) microscopy, two surface characterization techniques that precisely locate the height of fluorescent objects relative to the silicon surface with nm resolution. Freestanding lipid bilayers are formed by rupture of lipid vesicles and are stable for over 24 hrs. We investigate the structure-function relationship of the voltage-gated potassium channel KvAP. KvAP overexpressed in E. Coli is detergent-free extracted and purified using polymer nanodiscs. We show reconstitution of functional KvAP in black lipid membranes and in freestanding lipid bilayers on the membrane interferometer. KvAP channels are mutated and tagged with a fluorescent label at different positions at the S3 or S4 strand. Progress on electrical and FLIC measurements of fluorescently labeled KvAP will be described.

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