Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid
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 combi...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2023-01-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/85836 |
| _version_ | 1811165074541248512 |
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| author | Esteban Suárez-Delgado Maru Orozco-Contreras Gisela E Rangel-Yescas Leon D Islas |
| author_facet | Esteban Suárez-Delgado Maru Orozco-Contreras Gisela E Rangel-Yescas Leon D Islas |
| author_sort | Esteban Suárez-Delgado |
| collection | DOAJ |
| description | 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 (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 reveal the existence of a very slow transition in the deactivation 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. |
| first_indexed | 2024-04-10T15:31:38Z |
| format | Article |
| id | doaj.art-c67ca2a68a6446619f535cfb7488b1c5 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-10T15:31:38Z |
| publishDate | 2023-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-c67ca2a68a6446619f535cfb7488b1c52023-02-13T16:25:34ZengeLife Sciences Publications LtdeLife2050-084X2023-01-011210.7554/eLife.85836Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acidEsteban Suárez-Delgado0https://orcid.org/0000-0003-0147-3451Maru Orozco-Contreras1https://orcid.org/0000-0003-3315-4308Gisela E Rangel-Yescas2Leon D Islas3https://orcid.org/0000-0002-7461-5214Department of Physiology, School of Medicine, Universidad Nacional Autónoma de México, México City, MexicoDepartment of Physiology, School of Medicine, Universidad Nacional Autónoma de México, México City, MexicoDepartment of Physiology, School of Medicine, Universidad Nacional Autónoma de México, México City, MexicoDepartment of Physiology, School of Medicine, Universidad Nacional Autónoma de México, México City, MexicoVoltage-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 (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 reveal the existence of a very slow transition in the deactivation 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.https://elifesciences.org/articles/85836proton channelsfluorescencenon-canonical amino acidschannel gatingHEK293 cells |
| spellingShingle | Esteban Suárez-Delgado Maru Orozco-Contreras Gisela E Rangel-Yescas Leon D Islas Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid eLife proton channels fluorescence non-canonical amino acids channel gating HEK293 cells |
| title | Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid |
| title_full | Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid |
| title_fullStr | Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid |
| title_full_unstemmed | Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid |
| title_short | Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid |
| title_sort | activation pathway transitions in human voltage gated proton channels revealed by a non canonical fluorescent amino acid |
| topic | proton channels fluorescence non-canonical amino acids channel gating HEK293 cells |
| url | https://elifesciences.org/articles/85836 |
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