Human PIEZO1 Ion Channel Functions as a Split Protein.
PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4786117?pdf=render |
| _version_ | 1818475603958956032 |
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| author | Chilman Bae Thomas M Suchyna Lynn Ziegler Frederick Sachs Philip A Gottlieb |
| author_facet | Chilman Bae Thomas M Suchyna Lynn Ziegler Frederick Sachs Philip A Gottlieb |
| author_sort | Chilman Bae |
| collection | DOAJ |
| description | PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the "split protein" is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone. |
| first_indexed | 2024-12-10T09:14:50Z |
| format | Article |
| id | doaj.art-cb3209a747334edea5cc65bc15f82598 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T09:14:50Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-cb3209a747334edea5cc65bc15f825982022-12-22T01:54:53ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01113e015128910.1371/journal.pone.0151289Human PIEZO1 Ion Channel Functions as a Split Protein.Chilman BaeThomas M SuchynaLynn ZieglerFrederick SachsPhilip A GottliebPIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the "split protein" is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone.http://europepmc.org/articles/PMC4786117?pdf=render |
| spellingShingle | Chilman Bae Thomas M Suchyna Lynn Ziegler Frederick Sachs Philip A Gottlieb Human PIEZO1 Ion Channel Functions as a Split Protein. PLoS ONE |
| title | Human PIEZO1 Ion Channel Functions as a Split Protein. |
| title_full | Human PIEZO1 Ion Channel Functions as a Split Protein. |
| title_fullStr | Human PIEZO1 Ion Channel Functions as a Split Protein. |
| title_full_unstemmed | Human PIEZO1 Ion Channel Functions as a Split Protein. |
| title_short | Human PIEZO1 Ion Channel Functions as a Split Protein. |
| title_sort | human piezo1 ion channel functions as a split protein |
| url | http://europepmc.org/articles/PMC4786117?pdf=render |
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