Recent progress in genetically encoded voltage indicators (GEVIs) has led to several classes of proteins, which robustly report action potentials (APs) in cultured neurons. Thus, there is a need for fast, sensitive, bright and spectrally tunable reporters of membrane voltage. Membrane voltage is dynamically regulated in bacteria 3, fungi 4, plants 5 and many cell types and subcellular organelles in the human body, and is dysregulated in states of neuronal, cardiac and metabolic diseases. This bioelectric modulation is most famously observed in voltage-gated ion channels in neurons and cardiomyocytes, but voltage also affects the activity of G protein-coupled receptors and some transmembrane enzymes 2.
#Semaphor light t5 change voltage free
Membrane voltage acts on all transmembrane proteins: the membrane electric field pulls on charged residues, shifting the free energy landscape for charge-displacing conformational transitions 1. The freedom to choose a voltage indicator from an array of colours facilitates multicolour voltage imaging, as well as combination with other optical reporters and optogenetic actuators. Through a library screen, we identify linkers and fluorescent protein combinations that report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. A fluorescent protein is fused to an archaerhodopsin-derived voltage sensor. We present a palette of multicoloured brightly fluorescent genetically encoded voltage indicators with sensitivities from 8–13% Δ F/ F per 100 mV, and half-maximal response times from 4–7 ms. Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means.
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