Cargando…
Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4
When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (ΔF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanil...
Autores principales: | , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
2000
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217195/ https://www.ncbi.nlm.nih.gov/pubmed/10653897 |
_version_ | 1782149229279444992 |
---|---|
author | Sørensen, J.B. Cha, A. Latorre, R. Rosenman, E. Bezanilla, F. |
author_facet | Sørensen, J.B. Cha, A. Latorre, R. Rosenman, E. Bezanilla, F. |
author_sort | Sørensen, J.B. |
collection | PubMed |
description | When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (ΔF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391–408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3–S4 linker. In the deletion mutant, the maximal ΔF/F seen was diminished 10-fold, and the ΔF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3–S4 linker. The residual ΔF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of −90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a ΔF at extreme hyperpolarizations (more negative than −90 mV) only. A signal from the interaction with this group in the wt S3–S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic ΔF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment. |
format | Text |
id | pubmed-2217195 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2000 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22171952008-04-21 Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 Sørensen, J.B. Cha, A. Latorre, R. Rosenman, E. Bezanilla, F. J Gen Physiol Original Article When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (ΔF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391–408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3–S4 linker. In the deletion mutant, the maximal ΔF/F seen was diminished 10-fold, and the ΔF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3–S4 linker. The residual ΔF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of −90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a ΔF at extreme hyperpolarizations (more negative than −90 mV) only. A signal from the interaction with this group in the wt S3–S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic ΔF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment. The Rockefeller University Press 2000-02-01 /pmc/articles/PMC2217195/ /pubmed/10653897 Text en © 2000 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Sørensen, J.B. Cha, A. Latorre, R. Rosenman, E. Bezanilla, F. Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 |
title | Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 |
title_full | Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 |
title_fullStr | Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 |
title_full_unstemmed | Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 |
title_short | Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4 |
title_sort | deletion of the s3–s4 linker in theshaker potassium channel reveals two quenching groups near the outside of s4 |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217195/ https://www.ncbi.nlm.nih.gov/pubmed/10653897 |
work_keys_str_mv | AT sørensenjb deletionofthes3s4linkerintheshakerpotassiumchannelrevealstwoquenchinggroupsneartheoutsideofs4 AT chaa deletionofthes3s4linkerintheshakerpotassiumchannelrevealstwoquenchinggroupsneartheoutsideofs4 AT latorrer deletionofthes3s4linkerintheshakerpotassiumchannelrevealstwoquenchinggroupsneartheoutsideofs4 AT rosenmane deletionofthes3s4linkerintheshakerpotassiumchannelrevealstwoquenchinggroupsneartheoutsideofs4 AT bezanillaf deletionofthes3s4linkerintheshakerpotassiumchannelrevealstwoquenchinggroupsneartheoutsideofs4 |