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Specific effects of KChIP3/calsenilin/DREAM, but not KChIPs 1, 2 and 4, on calcium signalling and regulated secretion in PC12 cells.

Venn N, Haynes LP, Burgoyne RD - Biochem. J. (2008)

Bottom Line: KChIPs 1-4 were found to stimulate the traffic of Kv4.2 channels to the plasma membrane.In addition, KChIP3 but not KChIPs 1, 2 and 4 modified the ATP-induced Ca2+ signal resulting in a delay in recovery after the peak Ca2+ elevation and also specifically resulted in down-regulation of the Na+/Ca2+ exchanger NCX3, which could explain the effects on the Ca2+ signal and secretion.These results reveal a new role for KChIP3 in the regulation of Ca2+-regulated secretion and also suggest that the functions of each of the KChIPs may be more specialized than previously appreciated.

View Article: PubMed Central - PubMed

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

ABSTRACT
The KChIPs (K+ channel-interacting proteins) are members of the NCS (neuronal calcium sensor) protein family of Ca2+-binding proteins. It is unclear to what extent the KChIPs have distinct functions although they all interact with Kv4 K+ channels. KChIP3 has also been shown to repress transcription of specific genes via binding to DRE (downstream regulatory element) motifs and all KChIPs may share this function. In the present study, we have compared the function of isoforms of the four KChIPs. KChIPs 1-4 were found to stimulate the traffic of Kv4.2 channels to the plasma membrane. KChIP3 expression in PC12 cells resulted in an increase in exocytosis evoked by activation of purinergic receptors. In contrast, KChIPs 1, 2 and 4, although expressed to the same extent, had no effect on secretion. In addition, KChIP3 but not KChIPs 1, 2 and 4 modified the ATP-induced Ca2+ signal resulting in a delay in recovery after the peak Ca2+ elevation and also specifically resulted in down-regulation of the Na+/Ca2+ exchanger NCX3, which could explain the effects on the Ca2+ signal and secretion. Regulation of NCX3 by KChIP3 has been shown to occur via its DREAM (DRE antagonist modulator) function [Gomez-Villafuertes, Torres, Barrio, Savignac, Gabellini, Rizzato, Pintado, Gutierrez-Adan, Mellstrom, Carafoli and Naranjo (2005) J. Neurosci. 25, 10822-10830] suggesting that this activity might depend on the cellular context of expression of the various KChIPs. These results reveal a new role for KChIP3 in the regulation of Ca2+-regulated secretion and also suggest that the functions of each of the KChIPs may be more specialized than previously appreciated.

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Co-expression of KChIPs 1–4 with Kv4.2 in COS-7 cells stimulates traffic of Kv4.2 to the plasma membrane(A) COS-7 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and expression of the proteins was detected by Western blotting with an anti-GFP antibody. (B) COS-7 cells were transfected to express Kv4.2 and 48 h after transfection the cells were fixed and channel localization was detected by immunostaining with an anti-Kv4.2 antibody. (C–F) COS-7 cells were transfected to co-express Kv4.2, one of the KChIP–ECFPs, as indicated and channel localization was detected by immunostaining with anti-Kv4.2. The colour overlays show the KChIP–ECFPs in green and Kv4.2 in red, with co-localization seen in yellow. In all cases, traffic of the Kv4.2 to the plasma membrane was evident (arrowheads) along with KChIP localization to the plasma membrane. The scale bar represents 10 μm.
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Figure 1: Co-expression of KChIPs 1–4 with Kv4.2 in COS-7 cells stimulates traffic of Kv4.2 to the plasma membrane(A) COS-7 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and expression of the proteins was detected by Western blotting with an anti-GFP antibody. (B) COS-7 cells were transfected to express Kv4.2 and 48 h after transfection the cells were fixed and channel localization was detected by immunostaining with an anti-Kv4.2 antibody. (C–F) COS-7 cells were transfected to co-express Kv4.2, one of the KChIP–ECFPs, as indicated and channel localization was detected by immunostaining with anti-Kv4.2. The colour overlays show the KChIP–ECFPs in green and Kv4.2 in red, with co-localization seen in yellow. In all cases, traffic of the Kv4.2 to the plasma membrane was evident (arrowheads) along with KChIP localization to the plasma membrane. The scale bar represents 10 μm.

Mentions: Plasmids were constructed encoding four KChIP isoforms (KChIP1.2, KChIP2.3, KChIP3.1 and KChIP4.1) with each KChIP prepared as a C-terminal ECFP- or N-terminal FLAG-tagged construct. Since the terminology used for KChIP isoforms in the literature is inconsistent we have defined these by their accession number in the Materials and methods section but will refer to them henceforth as KChIPs 1–4 for simplicity. In order to validate the functionality of the constructs and to confirm that the fluorescently tagged KChIPs behaved as expected for the wild-type proteins, we assessed their expression in cell lines and their ability to stimulate the traffic of Kv4.2 to the plasma membrane based on imaging of expressed Kv4.2 [15]. In initial studies using HeLa cells, we successfully found cells expressing KChIPs 1, 2 or 4 but found poor survival of cells expressing KChIP3–ECFP, consistent with the reported apoptotic effect of KChIP3 expression in HeLa cells [27]. In contrast, all four KChIPs were expressed to the same extent in COS-7 cells (Figure 1A) and did not appear to affect cell survival. The four KChIPs expressed as ECFP fusion proteins had differing intracellular localizations consistent with previous results [14] with KChIP1 present on intracellular punctate structures as seen previously [15], KChIP2 being plasma-membrane-associated and KChIPs 3 and 4 being diffusely distributed throughout the cells (results not shown). As reported previously [15] Kv4.2, when expressed alone, trafficked poorly to the plasma membrane and accumulated in the Golgi complex (Figure 1B). In cells co-transfected to express Kv4.2 along with one of the KChIP proteins, all of the KChIPs co-localized with Kv4.2 and importantly all four KChIPs stimulated the traffic of the channel to the plasma membrane with all of the KChIPs also being associated with the plasma membrane (Figure 1), demonstrating a similar functional ability of the four KChIP isoforms studied to interact with Kv4.2 as seen in other studies [6,14].


Specific effects of KChIP3/calsenilin/DREAM, but not KChIPs 1, 2 and 4, on calcium signalling and regulated secretion in PC12 cells.

Venn N, Haynes LP, Burgoyne RD - Biochem. J. (2008)

Co-expression of KChIPs 1–4 with Kv4.2 in COS-7 cells stimulates traffic of Kv4.2 to the plasma membrane(A) COS-7 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and expression of the proteins was detected by Western blotting with an anti-GFP antibody. (B) COS-7 cells were transfected to express Kv4.2 and 48 h after transfection the cells were fixed and channel localization was detected by immunostaining with an anti-Kv4.2 antibody. (C–F) COS-7 cells were transfected to co-express Kv4.2, one of the KChIP–ECFPs, as indicated and channel localization was detected by immunostaining with anti-Kv4.2. The colour overlays show the KChIP–ECFPs in green and Kv4.2 in red, with co-localization seen in yellow. In all cases, traffic of the Kv4.2 to the plasma membrane was evident (arrowheads) along with KChIP localization to the plasma membrane. The scale bar represents 10 μm.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: Co-expression of KChIPs 1–4 with Kv4.2 in COS-7 cells stimulates traffic of Kv4.2 to the plasma membrane(A) COS-7 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and expression of the proteins was detected by Western blotting with an anti-GFP antibody. (B) COS-7 cells were transfected to express Kv4.2 and 48 h after transfection the cells were fixed and channel localization was detected by immunostaining with an anti-Kv4.2 antibody. (C–F) COS-7 cells were transfected to co-express Kv4.2, one of the KChIP–ECFPs, as indicated and channel localization was detected by immunostaining with anti-Kv4.2. The colour overlays show the KChIP–ECFPs in green and Kv4.2 in red, with co-localization seen in yellow. In all cases, traffic of the Kv4.2 to the plasma membrane was evident (arrowheads) along with KChIP localization to the plasma membrane. The scale bar represents 10 μm.
Mentions: Plasmids were constructed encoding four KChIP isoforms (KChIP1.2, KChIP2.3, KChIP3.1 and KChIP4.1) with each KChIP prepared as a C-terminal ECFP- or N-terminal FLAG-tagged construct. Since the terminology used for KChIP isoforms in the literature is inconsistent we have defined these by their accession number in the Materials and methods section but will refer to them henceforth as KChIPs 1–4 for simplicity. In order to validate the functionality of the constructs and to confirm that the fluorescently tagged KChIPs behaved as expected for the wild-type proteins, we assessed their expression in cell lines and their ability to stimulate the traffic of Kv4.2 to the plasma membrane based on imaging of expressed Kv4.2 [15]. In initial studies using HeLa cells, we successfully found cells expressing KChIPs 1, 2 or 4 but found poor survival of cells expressing KChIP3–ECFP, consistent with the reported apoptotic effect of KChIP3 expression in HeLa cells [27]. In contrast, all four KChIPs were expressed to the same extent in COS-7 cells (Figure 1A) and did not appear to affect cell survival. The four KChIPs expressed as ECFP fusion proteins had differing intracellular localizations consistent with previous results [14] with KChIP1 present on intracellular punctate structures as seen previously [15], KChIP2 being plasma-membrane-associated and KChIPs 3 and 4 being diffusely distributed throughout the cells (results not shown). As reported previously [15] Kv4.2, when expressed alone, trafficked poorly to the plasma membrane and accumulated in the Golgi complex (Figure 1B). In cells co-transfected to express Kv4.2 along with one of the KChIP proteins, all of the KChIPs co-localized with Kv4.2 and importantly all four KChIPs stimulated the traffic of the channel to the plasma membrane with all of the KChIPs also being associated with the plasma membrane (Figure 1), demonstrating a similar functional ability of the four KChIP isoforms studied to interact with Kv4.2 as seen in other studies [6,14].

Bottom Line: KChIPs 1-4 were found to stimulate the traffic of Kv4.2 channels to the plasma membrane.In addition, KChIP3 but not KChIPs 1, 2 and 4 modified the ATP-induced Ca2+ signal resulting in a delay in recovery after the peak Ca2+ elevation and also specifically resulted in down-regulation of the Na+/Ca2+ exchanger NCX3, which could explain the effects on the Ca2+ signal and secretion.These results reveal a new role for KChIP3 in the regulation of Ca2+-regulated secretion and also suggest that the functions of each of the KChIPs may be more specialized than previously appreciated.

View Article: PubMed Central - PubMed

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

ABSTRACT
The KChIPs (K+ channel-interacting proteins) are members of the NCS (neuronal calcium sensor) protein family of Ca2+-binding proteins. It is unclear to what extent the KChIPs have distinct functions although they all interact with Kv4 K+ channels. KChIP3 has also been shown to repress transcription of specific genes via binding to DRE (downstream regulatory element) motifs and all KChIPs may share this function. In the present study, we have compared the function of isoforms of the four KChIPs. KChIPs 1-4 were found to stimulate the traffic of Kv4.2 channels to the plasma membrane. KChIP3 expression in PC12 cells resulted in an increase in exocytosis evoked by activation of purinergic receptors. In contrast, KChIPs 1, 2 and 4, although expressed to the same extent, had no effect on secretion. In addition, KChIP3 but not KChIPs 1, 2 and 4 modified the ATP-induced Ca2+ signal resulting in a delay in recovery after the peak Ca2+ elevation and also specifically resulted in down-regulation of the Na+/Ca2+ exchanger NCX3, which could explain the effects on the Ca2+ signal and secretion. Regulation of NCX3 by KChIP3 has been shown to occur via its DREAM (DRE antagonist modulator) function [Gomez-Villafuertes, Torres, Barrio, Savignac, Gabellini, Rizzato, Pintado, Gutierrez-Adan, Mellstrom, Carafoli and Naranjo (2005) J. Neurosci. 25, 10822-10830] suggesting that this activity might depend on the cellular context of expression of the various KChIPs. These results reveal a new role for KChIP3 in the regulation of Ca2+-regulated secretion and also suggest that the functions of each of the KChIPs may be more specialized than previously appreciated.

Show MeSH