PT  - JOURNAL ARTICLE
AU  - Parashar Thapa
AU  - Robert Stewart
AU  - Rebecka J. Sepela
AU  - Oscar Vivas
AU  - Laxmi K. Parajuli
AU  - Mark Lillya
AU  - Sebastian Fletcher-Taylor
AU  - Bruce E. Cohen
AU  - Karen Zito
AU  - Jon T. Sack
TI  - An imaging method to measure conformational changes of voltage sensors of endogenous ion channels in tissue
AID  - 10.1101/541805
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 541805
4099  - http://biorxiv.org/content/early/2019/02/10/541805.short
4100  - http://biorxiv.org/content/early/2019/02/10/541805.full
AB  - A primary goal of molecular physiology is to understand the coupling between protein conformational change and systemic function. To see the conformational changes of a voltage-sensing protein within tissue, we synthesized a fluorescent molecular probe compatible with two-photon microscopy and developed a method to deconvolve conformational changes from fluorescence images. The probe was a fluorescently-tagged variant of a tarantula venom peptide that binds Kv2-type voltage gated K+ channel proteins when their voltage-sensing domains are in a resting conformation. Kv2 proteins in cell membranes were labeled by the probe, and the intensity of labeling responded to voltage changes. Voltage-response characteristics were used to calibrate a statistical thermodynamic model relating labeling intensity to the conformations adopted by unlabeled voltage sensors. Two-photon imaging of rat brain slices revealed fluorescence consistent with conformation-selective labeling of endogenous neuronal Kv2 proteins. In principle, this method of deconvolving images into measures of protein conformational change is generalizable to other proteins labeled with conformation-selective probes.