Abstract
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.