Genetically encoded fluorescent voltage sensors using the voltage-sensing domain of Nematostella and Danio phosphatases exhibit fast kinetics.
A substantial increase in the speed of the optical response of genetically encoded fluorescent protein voltage sensors (FP voltage sensors) was achieved by using the voltage-sensing phosphatase genes of Nematostella vectensis and Danio rerio. A potential N. vectensis voltage-sensing phosphatase was identified in silico. The voltage-sensing domain (S1-S4) of the N. vectensis homolog was used to create an FP voltage sensor called Nema. By replacing the phosphatase with a cerulean/citrine FRET pair, a new FP voltage sensor was synthesized with fast off kinetics (Tau(off)<5ms). However, the signal was small (ΔF/F=0.4%/200mV). FP voltage sensors using the D. rerio voltage-sensing phosphatase homolog, designated Zahra and Zahra 2, exhibited fast on and off kinetics within 2ms of the time constants observed with the organic voltage-sensitive dye, di4-ANEPPS. Mutagenesis of the S4 region of the Danio FP voltage sensor shifted the voltage dependence to more negative potentials but did not noticeably affect the kinetics of the optical signal.
Center for Functional Connectomics, Korean Institute of Science and Technology, Seoul, Republic of Korea. bradbaker@kist.re.kr
Bradley J
BJ
Baker
Lei
L
Jin
Zhou
Z
Han
Lawrence B
LB
Cohen
Marko
M
Popovic
Jelena
J
Platisa
Vincent
V
Pieribone
D000200:Action Potentials / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation / Q000235:genetics; D005456:Fluorescent Dyes / Q000494:pharmacology; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D018408:Patch-Clamp Techniques / Q000379:methods; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012615:Sea Anemones / Q000201:enzymology / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D010744:Phosphoric Monoester Hydrolases