ANAP: A versatile, fluorescent probe of ion channel gating and regulation

Puljung, Michael C.

doi:10.1016/bs.mie.2021.01.048

Cited by 12 publications

(15 citation statements)

References 58 publications

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“…To study voltage-dependent transitions in a voltage sensor using PCF, it is desirable that the introduced fluorescent probe does not produce a major structural perturbation of the target protein. The relatively recently developed probe Anap (3-[6-acetylnaphthalen-2-ylamino]–2-aminopropanoic acid) is a small non-canonical fluorescent amino acid ( Figure 1B ), which has been shown to be easily genetically encoded in proteins expressed in eukaryotic cells ( Chatterjee et al, 2013 ; Puljung, 2021 ). Moreover, Anap has been successfully used as a reporter of voltage-dependent conformational changes ( Kalstrup and Blunck, 2013 ) and as a Fluorescence Resonance Energy Transfer (FRET) pair ( Gordon et al, 2018 ) to probe ion channel dynamics.…”

Section: Resultsmentioning

confidence: 99%

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Suárez-Delgado

Orozco-Contreras

Rangel‐Yescas

et al. 2023

eLife

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Voltage-dependent gating of the voltage-gated proton channels (HV1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents. To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependence on voltage and the pH gradient (DpH). We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix, and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization. The kinetics of the fluorescence signal reveals the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by DpH. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. These results shed light on the complexities of voltage-dependent opening of human HV1 channels.

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Section: Resultsmentioning

confidence: 99%

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Suárez-Delgado

Orozco-Contreras

Rangel‐Yescas

et al. 2023

eLife

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“…To study voltage-dependent transitions in a voltage sensor using patch-clamp fluorometry, it is desirable that the introduced fluorescent probe does not produce a major structural perturbation of the target protein. The relatively recently developed probe Anap (3-(6-acetylnaphtalen-2-ylamino)-2-aminopropanoic acid) is a small non-canonical fluorescent amino acid (Figure 1B), which has been shown to be easily genetically-encoded in proteins expressed in eukaryotic cells (Chatterjee et al, 2013; Puljung, 2021) Moreover, Anap has been successfully used as a reporter of voltage-dependent conformational changes (Kalstrup and Blunck, 2013) and as a FRET pair (Gordon et al, 2018) to probe ion channel dynamics. In this study, Anap was inserted into specific positions of the S4 segment of the human H V 1 proton channel (hH V 1) sequence (Figure 1A), with the purpose of examining its voltage and pH-dependent dynamics.…”

Section: Resultsmentioning

confidence: 99%

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Suárez-Delgado

Rangel‐Yescas

Islas

2022

Preprint

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Voltage-dependent gating of the voltage-gated proton channels (HV1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents. To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependance on voltage and the pH gradient (ΔpH). We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization. The kinetics of the fluorescence signal reveals the existence of a very slow transition in the activation pathway, which seems to be singularly regulated by ΔpH. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. These results shed light on the complexities of voltage-dependent opening of human HV1 channels.

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“…Protein labeling can be performed either through fluorescent amino acids (fAAs), which were directly incorporated into POI, or applying the UAA bearing a clickable moiety can be site‐specifically conjugated alternative fluorophores [73] . In the direct labeling strategy, proteins can be incorporated in fAAs such as a coumarin‐derived amino acid (CouAA), [74] acridonylalanine (ACD), [75] (acetylnaphthalenyl) amino propanoic acid (Anap), [76] dansylalanine [77] and TerphenylA [78] (Figure 5b). AnapRS/tRNA is necessary for the mammalian system to be able to encode the Anap protein, which undergoes a significant shift in emission maxima and fluorescence intensity depending on the circumstances of its surrounding environment [79] .…”

Section: Unnatural Amino Acids Systemmentioning

confidence: 99%

Chemical tags and beyond: Live‐cell protein labeling technologies for modern optical imaging

Saimi,

Chen

2023

Smart Molecules

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Imaging proteins with high resolution is crucial for studying cellular physiology and pathology. Fluorescence imaging is a privileged method to visualize proteins with subcellular precision in live cells. In recent years, there has been a tremendous advance in the field of fluorescent dyes that are optically more sophisticated than genetically‐encodable fluorescent proteins. In this review, we aim to discuss modern bioconjugation methods to specifically incorporate these dyes into protein‐of‐interests. We focus on advances in live‐cell labeling strategies and fluorescent probes, especially the HaloTag, SNAP‐tag, TMP‐tag, and unnatural amino acid systems and their applications. These protein labeling methods, along with cutting‐edge dyes and novel microscopy methods, have become the infrastructure for biological research in the era of super‐resolution imaging.

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ANAP: A versatile, fluorescent probe of ion channel gating and regulation

Cited by 12 publications

References 58 publications

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid

Chemical tags and beyond: Live‐cell protein labeling technologies for modern optical imaging

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