Improved fluorescent probes for the measurement of rapid changes in membrane potential

Grinvald, Amiram; Hildesheim, Rina; Farber, Ira; Anglister, Lili

doi:10.1016/s0006-3495(82)84520-7

Cited by 253 publications

(173 citation statements)

References 22 publications

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“…The observed changes of the fluorescent dye may not be generated by transmembrane potentials but by changes of local electrical fields mainly on the basis of the mechanism of electrochromy Grinvald et al, 1982). Therefore this principle should also be applicable for SR membranes.…”

Section: Discussionmentioning

confidence: 99%

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Butscher

Roudná

Apell

1999

Journal of Membrane Biology

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Abstract.A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/ release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca 2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca 2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca 2+ ions in state E 1 with an apparent half-saturation concentration, K M of 600 nM. In state P-E 2 two K M values, 5 M and 2.2 mM, were determined and are in fair agreement with published data. From Ca 2+ titrations in buffers with various pH and from pH titrations in P-E 2 , it could be demonstrated that H + binding is electrogenic and that Ca 2+ and H + compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E 1 with unoccupied ion sites, independent of the buffer composition.

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Section: Discussionmentioning

confidence: 99%

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Butscher

Roudná

Apell

1999

Journal of Membrane Biology

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“…It was possible to average 100 trials with no noticeable change in action potential amplitude or waveform (Antic and Zecevic, 1995). Mammalian slice preparations have generally been found to be more sensitive to photodynamic injury than invertebrate preparations (Grinvald et al, 1982;Antic et al, 1999). Mitral cells, however, tolerated well the high-intensity excitation light during successive recording trials if the exposures were kept relatively short (100 -200 msec) and separated by dark intervals lasting several minutes.…”

Section: Methodsmentioning

confidence: 99%

Voltage Imaging from Dendrites of Mitral Cells: EPSP Attenuation and Spike Trigger Zones

Djurišić¹,

Antic²,

Chen³

et al. 2004

J. Neurosci.

118

133

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To obtain a more complete description of individual neurons, it is necessary to complement the electrical patch pipette measurements with technologies that permit a massive parallel recording from many sites on neuronal processes. This can be achieved by using voltage imaging with intracellular dyes. With this approach, we investigated the functional structure of a mitral cell, the principal output neuron in the rat olfactory bulb. The most significant finding concerns the characteristics of EPSPs at the synaptic sites and surprisingly small attenuation along the trunk of the primary dendrite. Also, the experiments were performed to determine the number, location, and stability of spike trigger zones, the excitability of terminal dendritic branches, and the pattern and nature of spike initiation and propagation in the primary and secondary dendrites. The results show that optical data can be used to deduce the amplitude and shape of the EPSPs evoked by olfactory nerve stimulation at the site of origin (glomerular tuft) and to determine its attenuation along the entire length of the primary dendrite. This attenuation corresponds to an unusually large mean apparent "length constant" of the primary dendrite. Furthermore, the images of spike trigger zones showed that an action potential can be initiated in three different compartments of the mitral cell: the soma-axon region, the primary dendrite trunk, and the terminal dendritic tuft, which appears to be fully excitable. Finally, secondary dendrites clearly support the active propagation of action potentials.

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“…However, C-2 reversed phase silica eluted with a gradient from 1% acetic acid in water to methanol yielded 130 mg blue solid (0.051 mmol, 23%). MALDI-TOF m/z 2531 (M+, 14%), 2575 (13), 2487 (13). Polydispersity of the PEG reagent resulted in a series of product peaks ranging from m/z 2004-3104 with 44 m/z increments.…”

Section: -[(E)-2-(5-{(e)-2-[4-(dibutylamino)phenyl]ethenyl}-thiophenmentioning

confidence: 99%

“…Many styryl dyes, including the series of RH and JPW dyes, have been synthesized with a range of excitation and emission wavelengths and a variety of voltage responses. 5,6,13,14 VSDs that have high quantum yields and produce a large change in fractional fluorescence (ΔF/F) are desireable because they generate adequate signals at lower dye concentrations. Recently, Salama et al reported a series of long-wavelength VSDs designed to report cardiac action potentials (APs) from deeper layers of the heart by incorporating a thienyl group in the polymethine bridge to maintain the rigidity of the styryl chromophore while extending the fluorescence wavelengths.…”

Section: Introductionmentioning

confidence: 99%

Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol

et al. 2007

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Long wavelength voltage-sensitive dyes (VSDs) called Pittsburgh (PGH) dyes were recently synthesized by coupling various heterocyclic groups to a styryl-thiophene intermediate forming extended, partially rigidized chromophores. Unlike most styryl VSDs, dyes with a sulfonic acid anchor directly attached to the chromophore showed no solvatochromic absorption shifts. The limited water solubility of many long wavelength VSDs requires the use of surfactants to transport the dye through aqueous media and effectively label biological membranes. Here, we tested the chemical substitution of the sulfonic acid moiety with polyethyleneglycol (PEG) chains ranging from MW 750 to 5000, to overcome the poor solubility of VSDs while retaining their properties as VSDs. The chemical synthesis of PGH dyes and their PEG derivatives are described. The PEGderivatives were soluble in aqueous solutions (> 1 mM) and still reported membrane potential changes. In frog and mouse hearts, the voltage sensitivity (ΔF/F per action potential) and spectral properties of PEG dyes were the same as the sulfonated analogs. Thus, the solubility of VSDs can be considerably improved with small polyethyleneglycol chains and can provide an effective approach to improve staining of excitable tissues and optical recordings of membrane potential.

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Improved fluorescent probes for the measurement of rapid changes in membrane potential

Cited by 253 publications

References 22 publications

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Voltage Imaging from Dendrites of Mitral Cells: EPSP Attenuation and Spike Trigger Zones

Enhanced aqueous solubility of long wavelength voltage-sensitive dyes by covalent attachment of polyethylene glycol

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