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Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway.

Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, Burgoyne RD - J. Cell Biol. (2005)

Bottom Line: Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP.The EF-hand mutant had no effect on general exocytic traffic.When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, England, UK.

ABSTRACT
The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

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Co-distribution of KChIP1-EYFP with Golgi structures in rat hippocampal neurons. Primary neuronal cultures were transfected to express KChIP1-EYFP (green) and Golgi-ECFP (red). The confocal images show the presence of both proteins in the cell body and at specific sites along the dendrites. B and C show higher magnifications of a dendrite; the arrows in B indicate the Golgi outposts. Bars, 10 μm (A and B) or 2 μm (C).
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fig6: Co-distribution of KChIP1-EYFP with Golgi structures in rat hippocampal neurons. Primary neuronal cultures were transfected to express KChIP1-EYFP (green) and Golgi-ECFP (red). The confocal images show the presence of both proteins in the cell body and at specific sites along the dendrites. B and C show higher magnifications of a dendrite; the arrows in B indicate the Golgi outposts. Bars, 10 μm (A and B) or 2 μm (C).

Mentions: We used HeLa cells in this study because these do not express KChIP proteins or Kv4 channels endogenously, and allow analysis of their individual targeting and trafficking. To test whether the same situation occurred in neurons we examined primary cultures of rat hippocampal neurons. Endogenous KChIP1 was found in punctate structures in neurons (Shibata et al., 2003), and KChIP1-EYFP was targeted to intracellular punctate structures in the cell body (Fig. S4, A and B; available at http://www.jcb.org/cgi/content/full/jcb.200506005/DC1) and was distributed along the dendrites (Fig. S4 C) in proximal and distal areas (>120 μm from the cell body). In contrast, the closely related NCS1-EYFP was found on the plasma membrane (Fig. S4, D and E), as seen in HeLa cells (O'Callaghan et al., 2002, 2003a). In dendrites, it did not show evidence of a punctate localization (Fig. S4 F). Hippocampal neurons were shown to possess satellite secretory pathways with ER exit sites and associated Golgi outposts distributed in dendritic processes a long distance from the cell body (Pierce et al., 2001; Horton and Ehlers, 2003; Aridor et al., 2004). We examined the relationship of KChIP1-containing structures to these outposts in hippocampal neurons that were cotransfected with KChIP1-EYFP and Golgi-targeted ECFP. Both proteins were expressed in the perikarya and were present in the dendrites (Fig. 6). As expected, KChIP1-EYFP did not overlap exactly with the Golgi marker along the dendrites, but showed a marked co-distribution so that essentially every Golgi outpost had an associated KChIP1-EYFP–labeled structure.


Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway.

Hasdemir B, Fitzgerald DJ, Prior IA, Tepikin AV, Burgoyne RD - J. Cell Biol. (2005)

Co-distribution of KChIP1-EYFP with Golgi structures in rat hippocampal neurons. Primary neuronal cultures were transfected to express KChIP1-EYFP (green) and Golgi-ECFP (red). The confocal images show the presence of both proteins in the cell body and at specific sites along the dendrites. B and C show higher magnifications of a dendrite; the arrows in B indicate the Golgi outposts. Bars, 10 μm (A and B) or 2 μm (C).
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Related In: Results  -  Collection

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fig6: Co-distribution of KChIP1-EYFP with Golgi structures in rat hippocampal neurons. Primary neuronal cultures were transfected to express KChIP1-EYFP (green) and Golgi-ECFP (red). The confocal images show the presence of both proteins in the cell body and at specific sites along the dendrites. B and C show higher magnifications of a dendrite; the arrows in B indicate the Golgi outposts. Bars, 10 μm (A and B) or 2 μm (C).
Mentions: We used HeLa cells in this study because these do not express KChIP proteins or Kv4 channels endogenously, and allow analysis of their individual targeting and trafficking. To test whether the same situation occurred in neurons we examined primary cultures of rat hippocampal neurons. Endogenous KChIP1 was found in punctate structures in neurons (Shibata et al., 2003), and KChIP1-EYFP was targeted to intracellular punctate structures in the cell body (Fig. S4, A and B; available at http://www.jcb.org/cgi/content/full/jcb.200506005/DC1) and was distributed along the dendrites (Fig. S4 C) in proximal and distal areas (>120 μm from the cell body). In contrast, the closely related NCS1-EYFP was found on the plasma membrane (Fig. S4, D and E), as seen in HeLa cells (O'Callaghan et al., 2002, 2003a). In dendrites, it did not show evidence of a punctate localization (Fig. S4 F). Hippocampal neurons were shown to possess satellite secretory pathways with ER exit sites and associated Golgi outposts distributed in dendritic processes a long distance from the cell body (Pierce et al., 2001; Horton and Ehlers, 2003; Aridor et al., 2004). We examined the relationship of KChIP1-containing structures to these outposts in hippocampal neurons that were cotransfected with KChIP1-EYFP and Golgi-targeted ECFP. Both proteins were expressed in the perikarya and were present in the dendrites (Fig. 6). As expected, KChIP1-EYFP did not overlap exactly with the Golgi marker along the dendrites, but showed a marked co-distribution so that essentially every Golgi outpost had an associated KChIP1-EYFP–labeled structure.

Bottom Line: Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP.The EF-hand mutant had no effect on general exocytic traffic.When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, England, UK.

ABSTRACT
The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

Show MeSH
Related in: MedlinePlus