Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology w...

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Autores principales:	Ryan Richards, Robert E Dempski
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/806a306bbc12445eb8e85303abd8b057
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spelling	oai:doaj.org-article:806a306bbc12445eb8e85303abd8b0572021-11-18T08:08:14ZRe-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.1932-620310.1371/journal.pone.0050018https://doaj.org/article/806a306bbc12445eb8e85303abd8b0572012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23185520/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.Ryan RichardsRobert E DempskiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 11, p e50018 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ryan Richards Robert E Dempski Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
description	Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.
format	article
author	Ryan Richards Robert E Dempski
author_facet	Ryan Richards Robert E Dempski
author_sort	Ryan Richards
title	Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
title_short	Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
title_full	Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
title_fullStr	Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
title_full_unstemmed	Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
title_sort	re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/806a306bbc12445eb8e85303abd8b057
work_keys_str_mv	AT ryanrichards reintroductionoftransmembraneserineresiduesreducetheminimumporediameterofchannelrhodopsin2 AT robertedempski reintroductionoftransmembraneserineresiduesreducetheminimumporediameterofchannelrhodopsin2
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Re-introduction of transmembrane serine residues reduce the minimum pore diameter of channelrhodopsin-2.

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