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Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a

BACKGROUND: Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous in...

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Autores principales: Spear, John M., Koborssy, Dolly Al, Schwartz, Austin B., Johnson, Adam J., Audhya, Anjon, Fadool, Debra A., Stagg, Scott M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497498/
https://www.ncbi.nlm.nih.gov/pubmed/26156069
http://dx.doi.org/10.1186/s12858-015-0045-6
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author Spear, John M.
Koborssy, Dolly Al
Schwartz, Austin B.
Johnson, Adam J.
Audhya, Anjon
Fadool, Debra A.
Stagg, Scott M.
author_facet Spear, John M.
Koborssy, Dolly Al
Schwartz, Austin B.
Johnson, Adam J.
Audhya, Anjon
Fadool, Debra A.
Stagg, Scott M.
author_sort Spear, John M.
collection PubMed
description BACKGROUND: Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3. RESULTS: We have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins. CONCLUSION: These results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12858-015-0045-6) contains supplementary material, which is available to authorized users.
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spelling pubmed-44974982015-07-10 Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a Spear, John M. Koborssy, Dolly Al Schwartz, Austin B. Johnson, Adam J. Audhya, Anjon Fadool, Debra A. Stagg, Scott M. BMC Biochem Research Article BACKGROUND: Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3. RESULTS: We have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins. CONCLUSION: These results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12858-015-0045-6) contains supplementary material, which is available to authorized users. BioMed Central 2015-07-10 /pmc/articles/PMC4497498/ /pubmed/26156069 http://dx.doi.org/10.1186/s12858-015-0045-6 Text en © Spear et al. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Spear, John M.
Koborssy, Dolly Al
Schwartz, Austin B.
Johnson, Adam J.
Audhya, Anjon
Fadool, Debra A.
Stagg, Scott M.
Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a
title Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a
title_full Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a
title_fullStr Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a
title_full_unstemmed Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a
title_short Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a
title_sort kv1.3 contains an alternative c-terminal er exit motif and is recruited into copii vesicles by sec24a
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497498/
https://www.ncbi.nlm.nih.gov/pubmed/26156069
http://dx.doi.org/10.1186/s12858-015-0045-6
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