Limits...
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.

Show MeSH

Related in: MedlinePlus

Ts045 VSVG-GFP and KChIP1-ECFP in the secretory pathway and effect of inhibiting traffic. HeLa cells were cotransfected with KChIP1-ECFP and ts045 VSVG-GFP and were incubated at 40°C overnight, which causes retention of ts045 VSVG-GFP in the ER. The temperature was reduced to 32°C and the cells were fixed after various time intervals as indicated in the images. At 40°C, ts045 VSVG-GFP is retained in the ER (A); upon the shift to permissive temperature for 5 min, it exits the ER and appears in punctate structures, which are likely to be post-ER vesicles (B). It then traffics to the Golgi apparatus over the next 60 min (C) and further to the plasma membrane in secretory vesicles over a 120-min period (D). The localization of KChIP1-ECFP and ts045 VSVG-GFP is shown individually and in color overlay (KChIP1-ECFP in red and ts045 VSVG-GFP in green) with colocalization seen in yellow (the arrows point at vesicles that overlap and appear in yellow). (E and F) HeLa cells were cotransfected with plasmids encoding pcDNA3-αSNAP(L294A) and VSVG-GFP (E) or KChIP1-ECFP (F) or were triple-transfected with all three constructs (G). αSNAP(L294A), which was visualized by immunostaining the cells with anti-αSNAP antibody and using Texas red–streptavidin, inhibits vesicular traffic throughout the secretory pathway. Ts045 VSVG-GFP was allowed to traffic at 32°C for 5 h before fixation of the cells. The localizations of the proteins are shown individually and in color overlay with colocalization seen in yellow. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171252&req=5

fig3: Ts045 VSVG-GFP and KChIP1-ECFP in the secretory pathway and effect of inhibiting traffic. HeLa cells were cotransfected with KChIP1-ECFP and ts045 VSVG-GFP and were incubated at 40°C overnight, which causes retention of ts045 VSVG-GFP in the ER. The temperature was reduced to 32°C and the cells were fixed after various time intervals as indicated in the images. At 40°C, ts045 VSVG-GFP is retained in the ER (A); upon the shift to permissive temperature for 5 min, it exits the ER and appears in punctate structures, which are likely to be post-ER vesicles (B). It then traffics to the Golgi apparatus over the next 60 min (C) and further to the plasma membrane in secretory vesicles over a 120-min period (D). The localization of KChIP1-ECFP and ts045 VSVG-GFP is shown individually and in color overlay (KChIP1-ECFP in red and ts045 VSVG-GFP in green) with colocalization seen in yellow (the arrows point at vesicles that overlap and appear in yellow). (E and F) HeLa cells were cotransfected with plasmids encoding pcDNA3-αSNAP(L294A) and VSVG-GFP (E) or KChIP1-ECFP (F) or were triple-transfected with all three constructs (G). αSNAP(L294A), which was visualized by immunostaining the cells with anti-αSNAP antibody and using Texas red–streptavidin, inhibits vesicular traffic throughout the secretory pathway. Ts045 VSVG-GFP was allowed to traffic at 32°C for 5 h before fixation of the cells. The localizations of the proteins are shown individually and in color overlay with colocalization seen in yellow. Bars, 10 μm.

Mentions: We compared KChIP1 with ts045 VSVG-GFP, as a marker of the constitutive secretory pathway (Presley et al., 1997). At a restrictive temperature of 40°C, ts045 VSVG-GFP has a folding defect that retains it in the ER (Griffiths et al., 1985; Beckers et al., 1987; Bergmann, 1989). Transfer to a permissive temperature of 32°C allows the protein to exit from the ER and traffic to the Golgi apparatus and then on to the plasma membrane. We examined the localization of ts045 VSVG-GFP and KChIP1-ECFP in cotransfected HeLa cells to determine whether ts045 VSVG-GFP colocalizes with KChIP1-ECFP during its traffic through the secretory pathway. Cotransfected HeLa cells were incubated overnight at 40°C. The next day, they were transferred to 32°C, and captured by fixation after various time intervals. Before the temperature reduction to 32°C (0 min), ts045 VSVG-GFP appeared in a reticular pattern as it was retained in the ER with no overlap visible with KChIP1-ECFP (Fig. 3 A). Within 5 min of the shift to 32°C, vesicular structures began to appear—potentially vesicles budding off the ER—and a small proportion colocalized with KChIP1-ECFP–labeled vesicles (Fig. 3 B). Upon a temperature shift to 32°C for 15 min, ts045 VSVG-GFP redistributed to a perinuclear Golgi-like region and no longer overlapped with KChIP1-ECFP. The same situation was observed at 30 min (not depicted) and 60 min (Fig. 3 C). After 120 min at 32°C, ts045 VSVG-GFP was found in numerous punctate structures and on the plasma membrane. The punctate structures, likely to be vesicles that were trafficking from the Golgi apparatus to the plasma membrane, showed no colocalization with KChIP1-ECFP–labeled vesicles (Fig. 3 D). By 180 min at 32°C, ts045 VSVG-GFP had reached the plasma membrane, with a component still in the Golgi apparatus; again, no colocalization was seen with KChIP1-ECFP (unpublished data). These findings demonstrate that KChIP1-ECFP–labeled vesicles are part of a post-ER, pre-Golgi traffic system and are distinct from the bulk of those that are involved in traffic of VSVG from the ER to the Golgi complex. The colocalization of KChIP1-ECFP with a small subset of vesicles that contains ts045 VSVG-GFP, within 5 min after a shift to permissive temperature, suggests that KChIP1 vesicles may be close to a subset of ER exit sites that is used by VSVG or that there may be a small overlap between the two pathways.


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)

Ts045 VSVG-GFP and KChIP1-ECFP in the secretory pathway and effect of inhibiting traffic. HeLa cells were cotransfected with KChIP1-ECFP and ts045 VSVG-GFP and were incubated at 40°C overnight, which causes retention of ts045 VSVG-GFP in the ER. The temperature was reduced to 32°C and the cells were fixed after various time intervals as indicated in the images. At 40°C, ts045 VSVG-GFP is retained in the ER (A); upon the shift to permissive temperature for 5 min, it exits the ER and appears in punctate structures, which are likely to be post-ER vesicles (B). It then traffics to the Golgi apparatus over the next 60 min (C) and further to the plasma membrane in secretory vesicles over a 120-min period (D). The localization of KChIP1-ECFP and ts045 VSVG-GFP is shown individually and in color overlay (KChIP1-ECFP in red and ts045 VSVG-GFP in green) with colocalization seen in yellow (the arrows point at vesicles that overlap and appear in yellow). (E and F) HeLa cells were cotransfected with plasmids encoding pcDNA3-αSNAP(L294A) and VSVG-GFP (E) or KChIP1-ECFP (F) or were triple-transfected with all three constructs (G). αSNAP(L294A), which was visualized by immunostaining the cells with anti-αSNAP antibody and using Texas red–streptavidin, inhibits vesicular traffic throughout the secretory pathway. Ts045 VSVG-GFP was allowed to traffic at 32°C for 5 h before fixation of the cells. The localizations of the proteins are shown individually and in color overlay with colocalization seen in yellow. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Ts045 VSVG-GFP and KChIP1-ECFP in the secretory pathway and effect of inhibiting traffic. HeLa cells were cotransfected with KChIP1-ECFP and ts045 VSVG-GFP and were incubated at 40°C overnight, which causes retention of ts045 VSVG-GFP in the ER. The temperature was reduced to 32°C and the cells were fixed after various time intervals as indicated in the images. At 40°C, ts045 VSVG-GFP is retained in the ER (A); upon the shift to permissive temperature for 5 min, it exits the ER and appears in punctate structures, which are likely to be post-ER vesicles (B). It then traffics to the Golgi apparatus over the next 60 min (C) and further to the plasma membrane in secretory vesicles over a 120-min period (D). The localization of KChIP1-ECFP and ts045 VSVG-GFP is shown individually and in color overlay (KChIP1-ECFP in red and ts045 VSVG-GFP in green) with colocalization seen in yellow (the arrows point at vesicles that overlap and appear in yellow). (E and F) HeLa cells were cotransfected with plasmids encoding pcDNA3-αSNAP(L294A) and VSVG-GFP (E) or KChIP1-ECFP (F) or were triple-transfected with all three constructs (G). αSNAP(L294A), which was visualized by immunostaining the cells with anti-αSNAP antibody and using Texas red–streptavidin, inhibits vesicular traffic throughout the secretory pathway. Ts045 VSVG-GFP was allowed to traffic at 32°C for 5 h before fixation of the cells. The localizations of the proteins are shown individually and in color overlay with colocalization seen in yellow. Bars, 10 μm.
Mentions: We compared KChIP1 with ts045 VSVG-GFP, as a marker of the constitutive secretory pathway (Presley et al., 1997). At a restrictive temperature of 40°C, ts045 VSVG-GFP has a folding defect that retains it in the ER (Griffiths et al., 1985; Beckers et al., 1987; Bergmann, 1989). Transfer to a permissive temperature of 32°C allows the protein to exit from the ER and traffic to the Golgi apparatus and then on to the plasma membrane. We examined the localization of ts045 VSVG-GFP and KChIP1-ECFP in cotransfected HeLa cells to determine whether ts045 VSVG-GFP colocalizes with KChIP1-ECFP during its traffic through the secretory pathway. Cotransfected HeLa cells were incubated overnight at 40°C. The next day, they were transferred to 32°C, and captured by fixation after various time intervals. Before the temperature reduction to 32°C (0 min), ts045 VSVG-GFP appeared in a reticular pattern as it was retained in the ER with no overlap visible with KChIP1-ECFP (Fig. 3 A). Within 5 min of the shift to 32°C, vesicular structures began to appear—potentially vesicles budding off the ER—and a small proportion colocalized with KChIP1-ECFP–labeled vesicles (Fig. 3 B). Upon a temperature shift to 32°C for 15 min, ts045 VSVG-GFP redistributed to a perinuclear Golgi-like region and no longer overlapped with KChIP1-ECFP. The same situation was observed at 30 min (not depicted) and 60 min (Fig. 3 C). After 120 min at 32°C, ts045 VSVG-GFP was found in numerous punctate structures and on the plasma membrane. The punctate structures, likely to be vesicles that were trafficking from the Golgi apparatus to the plasma membrane, showed no colocalization with KChIP1-ECFP–labeled vesicles (Fig. 3 D). By 180 min at 32°C, ts045 VSVG-GFP had reached the plasma membrane, with a component still in the Golgi apparatus; again, no colocalization was seen with KChIP1-ECFP (unpublished data). These findings demonstrate that KChIP1-ECFP–labeled vesicles are part of a post-ER, pre-Golgi traffic system and are distinct from the bulk of those that are involved in traffic of VSVG from the ER to the Golgi complex. The colocalization of KChIP1-ECFP with a small subset of vesicles that contains ts045 VSVG-GFP, within 5 min after a shift to permissive temperature, suggests that KChIP1 vesicles may be close to a subset of ER exit sites that is used by VSVG or that there may be a small overlap between the two pathways.

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