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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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The mobility of KChIP1-EYFP–labeled vesicles. HeLa cells were transfected to express KChIP1-EYFP and the dynamics of the KChIP1 vesicles were imaged by time-lapse confocal laser scanning microscopy at 20°C. (A) Movement of the vesicles is shown in an overlaid image of a cell at the beginning and 180 s later with the initial image in green and the later image in red. Stationary vesicles appear in yellow. Examples of vesicles showing directed stop-go motion are circled, with their direction of travel indicated by the arrows. (B) Image series show the movement of a KChIP1-EYFP–labeled vesicle in a control cell, with a neighboring one remaining stationary at this time interval (marked by arrow heads). (C) The mobility is not affected by the EF-hand mutation of KChIP1. (D) Incubation in 10 μM nocodazole for 1 h resulted in complete loss of the mobility of the KChIP1-EYFP–labeled structures. Bars, 10 μm (A) or 5 μm (B–D).
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fig2: The mobility of KChIP1-EYFP–labeled vesicles. HeLa cells were transfected to express KChIP1-EYFP and the dynamics of the KChIP1 vesicles were imaged by time-lapse confocal laser scanning microscopy at 20°C. (A) Movement of the vesicles is shown in an overlaid image of a cell at the beginning and 180 s later with the initial image in green and the later image in red. Stationary vesicles appear in yellow. Examples of vesicles showing directed stop-go motion are circled, with their direction of travel indicated by the arrows. (B) Image series show the movement of a KChIP1-EYFP–labeled vesicle in a control cell, with a neighboring one remaining stationary at this time interval (marked by arrow heads). (C) The mobility is not affected by the EF-hand mutation of KChIP1. (D) Incubation in 10 μM nocodazole for 1 h resulted in complete loss of the mobility of the KChIP1-EYFP–labeled structures. Bars, 10 μm (A) or 5 μm (B–D).

Mentions: KChIP1-EYFP expressed alone is targeted via its myristoyl tail to punctate structures, which do not colocalize completely with any known intracellular markers. However, they do overlap partially with ERGIC-53–positive structures. The minimal myristoylation motif, KChIP1(1–11)-EYFP, leads to progressive enlargement of the ERGIC because of blockade of traffic that is mediated by COPI, and then overlaps completely with ERGIC-53, β-COP, and ECFP-Kv4.2 (O'Callaghan et al., 2003a). Live cell imaging revealed that a proportion of the KChIP1-EYFP–labeled structures were mobile (Fig. 2, A and B). Vesicles within the perinuclear region essentially were stationary, but vesicles that are more peripheral showed random movement over short distances (<2 μm) or showed directed stop-go movement over distances ≤20 μm at rates of ≤0.15 μm/s. The directed movement occurred into and away from the perinuclear region (Fig. 2 A). The EF-hand mutant did not affect mobility of the vesicles (Fig. 2 C). Both types of movement were microtubule dependent, because incubation with nocodazole did not affect the morphology of the vesicles, but did cause them to become immobile (Fig. 2 D). These findings are consistent with the KChIP structures being transport vesicles.


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)

The mobility of KChIP1-EYFP–labeled vesicles. HeLa cells were transfected to express KChIP1-EYFP and the dynamics of the KChIP1 vesicles were imaged by time-lapse confocal laser scanning microscopy at 20°C. (A) Movement of the vesicles is shown in an overlaid image of a cell at the beginning and 180 s later with the initial image in green and the later image in red. Stationary vesicles appear in yellow. Examples of vesicles showing directed stop-go motion are circled, with their direction of travel indicated by the arrows. (B) Image series show the movement of a KChIP1-EYFP–labeled vesicle in a control cell, with a neighboring one remaining stationary at this time interval (marked by arrow heads). (C) The mobility is not affected by the EF-hand mutation of KChIP1. (D) Incubation in 10 μM nocodazole for 1 h resulted in complete loss of the mobility of the KChIP1-EYFP–labeled structures. Bars, 10 μm (A) or 5 μm (B–D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171252&req=5

fig2: The mobility of KChIP1-EYFP–labeled vesicles. HeLa cells were transfected to express KChIP1-EYFP and the dynamics of the KChIP1 vesicles were imaged by time-lapse confocal laser scanning microscopy at 20°C. (A) Movement of the vesicles is shown in an overlaid image of a cell at the beginning and 180 s later with the initial image in green and the later image in red. Stationary vesicles appear in yellow. Examples of vesicles showing directed stop-go motion are circled, with their direction of travel indicated by the arrows. (B) Image series show the movement of a KChIP1-EYFP–labeled vesicle in a control cell, with a neighboring one remaining stationary at this time interval (marked by arrow heads). (C) The mobility is not affected by the EF-hand mutation of KChIP1. (D) Incubation in 10 μM nocodazole for 1 h resulted in complete loss of the mobility of the KChIP1-EYFP–labeled structures. Bars, 10 μm (A) or 5 μm (B–D).
Mentions: KChIP1-EYFP expressed alone is targeted via its myristoyl tail to punctate structures, which do not colocalize completely with any known intracellular markers. However, they do overlap partially with ERGIC-53–positive structures. The minimal myristoylation motif, KChIP1(1–11)-EYFP, leads to progressive enlargement of the ERGIC because of blockade of traffic that is mediated by COPI, and then overlaps completely with ERGIC-53, β-COP, and ECFP-Kv4.2 (O'Callaghan et al., 2003a). Live cell imaging revealed that a proportion of the KChIP1-EYFP–labeled structures were mobile (Fig. 2, A and B). Vesicles within the perinuclear region essentially were stationary, but vesicles that are more peripheral showed random movement over short distances (<2 μm) or showed directed stop-go movement over distances ≤20 μm at rates of ≤0.15 μm/s. The directed movement occurred into and away from the perinuclear region (Fig. 2 A). The EF-hand mutant did not affect mobility of the vesicles (Fig. 2 C). Both types of movement were microtubule dependent, because incubation with nocodazole did not affect the morphology of the vesicles, but did cause them to become immobile (Fig. 2 D). These findings are consistent with the KChIP structures being transport vesicles.

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