Piao, Hong Hua Rajakumar, Dhanarajan Kang, Bok Eum Kim, Eun Ha Baker, Bradley J. 2024-01-20T08:01:37Z 2024-01-20T08:01:37Z 2021-09-05 2015-01-07 0270-6474 https://pubs.kist.re.kr/handle/201004/125876 ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1-S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil-coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics < 10 ms. Optimization of the linker sequence between S4 and the fluorescent protein resulted in a new ArcLight-derived probe, Bongwoori, capable of resolving action potentials in a hippocampal neuron firing at 60 Hz. Additional manipulation of the voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity. English SOC NEUROSCIENCE ELECTRICAL-ACTIVITY PROTEIN NEURONS CHANNEL PROBE ELECTROPHYSIOLOGY PHOSPHATASES KINETICS SEQUENCE Combinatorial Mutagenesis of the Voltage-Sensing Domain Enables the Optical Resolution of Action Potentials Firing at 60 Hz by a Genetically Encoded Fluorescent Sensor of Membrane Potential Article 10.1523/JNEUROSCI.3008-14.2015 1 JOURNAL OF NEUROSCIENCE, v.35, no.1, pp.372 - 385 JOURNAL OF NEUROSCIENCE 35 1 372 385 scie scopus 000349407200032 2-s2.0-84920517970 Neurosciences Neurosciences & Neurology Article ELECTRICAL-ACTIVITY PROTEIN NEURONS CHANNEL PROBE ELECTROPHYSIOLOGY PHOSPHATASES KINETICS SEQUENCE fluorescent protein omega current voltage imaging voltage sensor voltage-sensing domain voltage-sensing phosphatase