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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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Related in: MedlinePlus

Effects of the KChIP1 EF-hand mutant on membrane traffic. (A) In HeLa cells coexpressing KChIP1-EYFP, the Kv4.2 channel traffics to the plasma membrane. The KChIP1 EF-hand mutant (KChIP1(2–4)-EYFP) is targeted to the same vesicles as is KChIP1-ECFP (B), but in contrast to KChIP1-EYFP, it disrupts traffic of ECFP-Kv4.2 to the plasma membrane and traps the channel in vesicles (C). The EF-hand mutant does not disrupt traffic of VSVG-GFP to the plasma membrane over a 5-h period at permissive temperature (D). The color overlays show coexpressed proteins in red and green with colocalization appearing in yellow. Bars, 10 μm. Quantification of the effect of KChIP1-EYFP and KChIP1(2–4)-EYFP on ECFP-Kv4.2 traffic to the plasma membrane. (E) HeLa cells were transfected to express ECFP-Kv4.2 alone or in combination with KChIP1-EYFP or the EF-hand mutant KChIP1(2–4)-EYFP. ECFP-Kv4.2 fluorescence was imaged and quantified by drawing regions of interest around the outside and the inside of the plasma membrane (bottom panels) to allow determination of the percentage of total fluorescence at the plasma membrane. (F) Mean data derived from 25–29 cells expressing ECFP-Kv4.2 alone or coexpressing KChIP1-EYFP or KChIP1(2–4)-EYFP.
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fig1: Effects of the KChIP1 EF-hand mutant on membrane traffic. (A) In HeLa cells coexpressing KChIP1-EYFP, the Kv4.2 channel traffics to the plasma membrane. The KChIP1 EF-hand mutant (KChIP1(2–4)-EYFP) is targeted to the same vesicles as is KChIP1-ECFP (B), but in contrast to KChIP1-EYFP, it disrupts traffic of ECFP-Kv4.2 to the plasma membrane and traps the channel in vesicles (C). The EF-hand mutant does not disrupt traffic of VSVG-GFP to the plasma membrane over a 5-h period at permissive temperature (D). The color overlays show coexpressed proteins in red and green with colocalization appearing in yellow. Bars, 10 μm. Quantification of the effect of KChIP1-EYFP and KChIP1(2–4)-EYFP on ECFP-Kv4.2 traffic to the plasma membrane. (E) HeLa cells were transfected to express ECFP-Kv4.2 alone or in combination with KChIP1-EYFP or the EF-hand mutant KChIP1(2–4)-EYFP. ECFP-Kv4.2 fluorescence was imaged and quantified by drawing regions of interest around the outside and the inside of the plasma membrane (bottom panels) to allow determination of the percentage of total fluorescence at the plasma membrane. (F) Mean data derived from 25–29 cells expressing ECFP-Kv4.2 alone or coexpressing KChIP1-EYFP or KChIP1(2–4)-EYFP.

Mentions: Coexpression of Kv4.2 with KChIP1 results in a marked change in the intracellular localization of both proteins, with redistribution to the plasma membrane (Fig. 1 A). The ability of KChIP1 to traffic Kv4.2 beyond the Golgi complex must mean that the two proteins interact at some stage during ER-to-Golgi or intra-Golgi traffic. It was shown that the functional effects of KChIP1 on the Kv4.2 channel—including the increase in Kv4.2 current density, which was measured electrophysiologically—are Ca2+ dependent, whereas the interaction between the two proteins is not (An et al., 2000). It is not known why the Ca2+-binding ability of KChIP1 is essential and where in channel traffic the Ca2+-dependent step occurs. We used the same triple EF mutant of KChIP1 (KChIP1(2–4)-EYFP) as described in An et al. (2000), which could still bind to Kv4. The localization of KChIP1 itself was not affected by the EF-hand mutation, because KChIP1-ECFP and KChIP1(2–4)-EYFP overlapped in punctate structures in coexpressing cells (Fig. 1 B), and hence, the targeting of KChIP1 was not dependent on Ca2+. In contrast to wild-type KChIP1, the coexpression of the EF-hand mutant with ECFP-Kv4.2 did not allow traffic of the channel to the plasma membrane in any of the cells that were examined (Fig. 1 C). Surprisingly, the channel did not simply remain in the Golgi region as in cells that express Kv4.2 alone, but instead had a punctate distribution like KChIP1(2–4)-EYFP. In all cells that were examined, >50% of the punctate spots were positive for KChIP1(2–4)-EYFP and ECFP-Kv4.2 (see color overlay). The effect of KChIP1-EYFP and the EF-hand mutant on traffic of ECFP-Kv4.2 to the cell surface was quantified by determining the level of fluorescence at the cell periphery and in the cell as a whole (Fig. 1 E). Coexpression with KChIP1-EYFP resulted in a threefold increase in fluorescence at the cell surface. Coexpression with KChIP1(2–4)-EYFP did not increase the percentage of ECFP-Kv4.2 that reached the cell surface (Fig. 1 F). These data are consistent with previous electrophysiologic analyses (An et al., 2000). The EF-hand mutant did not affect membrane traffic in general, because vesicular stomatitis virus glycoprotein (VSVG)—expressed as ts045 VSVG-GFP (a well characterized protein for following constitutive exocytic vesicular traffic [Presley et al., 1997])—did not colocalize with KChIP1(2–4) and was able to traffic to the plasma membrane (Fig. 1 D).


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)

Effects of the KChIP1 EF-hand mutant on membrane traffic. (A) In HeLa cells coexpressing KChIP1-EYFP, the Kv4.2 channel traffics to the plasma membrane. The KChIP1 EF-hand mutant (KChIP1(2–4)-EYFP) is targeted to the same vesicles as is KChIP1-ECFP (B), but in contrast to KChIP1-EYFP, it disrupts traffic of ECFP-Kv4.2 to the plasma membrane and traps the channel in vesicles (C). The EF-hand mutant does not disrupt traffic of VSVG-GFP to the plasma membrane over a 5-h period at permissive temperature (D). The color overlays show coexpressed proteins in red and green with colocalization appearing in yellow. Bars, 10 μm. Quantification of the effect of KChIP1-EYFP and KChIP1(2–4)-EYFP on ECFP-Kv4.2 traffic to the plasma membrane. (E) HeLa cells were transfected to express ECFP-Kv4.2 alone or in combination with KChIP1-EYFP or the EF-hand mutant KChIP1(2–4)-EYFP. ECFP-Kv4.2 fluorescence was imaged and quantified by drawing regions of interest around the outside and the inside of the plasma membrane (bottom panels) to allow determination of the percentage of total fluorescence at the plasma membrane. (F) Mean data derived from 25–29 cells expressing ECFP-Kv4.2 alone or coexpressing KChIP1-EYFP or KChIP1(2–4)-EYFP.
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Related In: Results  -  Collection

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fig1: Effects of the KChIP1 EF-hand mutant on membrane traffic. (A) In HeLa cells coexpressing KChIP1-EYFP, the Kv4.2 channel traffics to the plasma membrane. The KChIP1 EF-hand mutant (KChIP1(2–4)-EYFP) is targeted to the same vesicles as is KChIP1-ECFP (B), but in contrast to KChIP1-EYFP, it disrupts traffic of ECFP-Kv4.2 to the plasma membrane and traps the channel in vesicles (C). The EF-hand mutant does not disrupt traffic of VSVG-GFP to the plasma membrane over a 5-h period at permissive temperature (D). The color overlays show coexpressed proteins in red and green with colocalization appearing in yellow. Bars, 10 μm. Quantification of the effect of KChIP1-EYFP and KChIP1(2–4)-EYFP on ECFP-Kv4.2 traffic to the plasma membrane. (E) HeLa cells were transfected to express ECFP-Kv4.2 alone or in combination with KChIP1-EYFP or the EF-hand mutant KChIP1(2–4)-EYFP. ECFP-Kv4.2 fluorescence was imaged and quantified by drawing regions of interest around the outside and the inside of the plasma membrane (bottom panels) to allow determination of the percentage of total fluorescence at the plasma membrane. (F) Mean data derived from 25–29 cells expressing ECFP-Kv4.2 alone or coexpressing KChIP1-EYFP or KChIP1(2–4)-EYFP.
Mentions: Coexpression of Kv4.2 with KChIP1 results in a marked change in the intracellular localization of both proteins, with redistribution to the plasma membrane (Fig. 1 A). The ability of KChIP1 to traffic Kv4.2 beyond the Golgi complex must mean that the two proteins interact at some stage during ER-to-Golgi or intra-Golgi traffic. It was shown that the functional effects of KChIP1 on the Kv4.2 channel—including the increase in Kv4.2 current density, which was measured electrophysiologically—are Ca2+ dependent, whereas the interaction between the two proteins is not (An et al., 2000). It is not known why the Ca2+-binding ability of KChIP1 is essential and where in channel traffic the Ca2+-dependent step occurs. We used the same triple EF mutant of KChIP1 (KChIP1(2–4)-EYFP) as described in An et al. (2000), which could still bind to Kv4. The localization of KChIP1 itself was not affected by the EF-hand mutation, because KChIP1-ECFP and KChIP1(2–4)-EYFP overlapped in punctate structures in coexpressing cells (Fig. 1 B), and hence, the targeting of KChIP1 was not dependent on Ca2+. In contrast to wild-type KChIP1, the coexpression of the EF-hand mutant with ECFP-Kv4.2 did not allow traffic of the channel to the plasma membrane in any of the cells that were examined (Fig. 1 C). Surprisingly, the channel did not simply remain in the Golgi region as in cells that express Kv4.2 alone, but instead had a punctate distribution like KChIP1(2–4)-EYFP. In all cells that were examined, >50% of the punctate spots were positive for KChIP1(2–4)-EYFP and ECFP-Kv4.2 (see color overlay). The effect of KChIP1-EYFP and the EF-hand mutant on traffic of ECFP-Kv4.2 to the cell surface was quantified by determining the level of fluorescence at the cell periphery and in the cell as a whole (Fig. 1 E). Coexpression with KChIP1-EYFP resulted in a threefold increase in fluorescence at the cell surface. Coexpression with KChIP1(2–4)-EYFP did not increase the percentage of ECFP-Kv4.2 that reached the cell surface (Fig. 1 F). These data are consistent with previous electrophysiologic analyses (An et al., 2000). The EF-hand mutant did not affect membrane traffic in general, because vesicular stomatitis virus glycoprotein (VSVG)—expressed as ts045 VSVG-GFP (a well characterized protein for following constitutive exocytic vesicular traffic [Presley et al., 1997])—did not colocalize with KChIP1(2–4) and was able to traffic to the plasma membrane (Fig. 1 D).

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