Format

Send to

Choose Destination
Biophys J. 2010 Oct 6;99(7):2355-65. doi: 10.1016/j.bpj.2010.07.037.

Improved probes for hybrid voltage sensor imaging.

Author information

1
Department of Physiology, University of Wisconsin, Madison, WI, USA.

Abstract

Hybrid voltage sensors (hVoS) probe membrane potential by coupling the fluorescence of membrane-anchored proteins to the movement of a membrane-embedded hydrophobic anion dipicrylamine. Fluorescence resonance energy transfer between these two components transduces voltage changes into fluorescence changes, providing a signal for imaging electrical activity in genetically targeted cells. To improve hVoS signals, we systematically varied the optical properties, membrane targeting motifs, and linkages of fluorescent proteins to optimize the normalized fluorescence change (ΔF/F) and signal/noise ratio. The best results were obtained with cerulean fluorescent protein tagged N-terminally with a GAP43 motif and C-terminally with a truncated h-ras motif. With 100 mV steps in PC12 cells, this probe produced ΔF/F = 26% (4 μM dipicrylamine), which was threefold greater than that obtained with the original farnesylated EGFP construct. We also obtained a fivefold greater signal/noise ratio, which was 70% of a theoretical optimum. We designate this GAP43-CerFP-t-h-ras construct as hVoS 2.0. With the aid of a theoretical analysis, we estimated that hVoS 2.0 places its fluorophore ∼40 Å from the bilayer midplane. hVoS 2.0 performed well in cultured hippocampal neurons, where single action potentials produced clear fluorescence changes in a single trial. This improved probe should help investigators image voltage in genetically targeted neurons.

PMID:
20923671
PMCID:
PMC3042572
DOI:
10.1016/j.bpj.2010.07.037
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center