Combining Electrical and Optical Measurements to Reveal the Structure-Function Relationship of Voltage-Gated Potassium Channels

Abstract

Although the structure of the voltage-gated potassium channel KvAP has been resolved more than a decade ago, there is still controversy about their precise gating mechanism. The discrepancies among proposed models result in part from differences in experimental techniques: electrical measurements of channels in lipid membranes to study their function versus determination of the structure of channels purified using detergents. To be able to combine functional and structural measurements, we developed a second-generation membrane interferometer in which both electrophysiology measurements and high-resolution fluorescence microscopy imaging can be performed on ion channels reconstituted in a lipid membrane, a much advanced design over the original (Ganesan et al., Proc. Natl. Acad. Sci. 106, 2008). 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 mirror with nanometer resolution. KvAP overexpressed in E. coli is detergent-free extracted using polymer nanodiscs. We show that functional KvAP can be reconstituted in black lipid membranes and freestanding lipid bilayers on the membrane interferometer. No differences in gating properties were found between KvAP extracted by polymer nanodiscs versus detergents. We label the channels with a fluorescent tag at different positions at the S3 or S4 strand. Our goal is to correlate the change in position of the S3 and S4 strands measured using FLIC microscopy with single-channel electrical measurements to reveal the structure-function relationship of KvAP. Progress towards this goal will be described.