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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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Expression of KChIP3, but not KChIPs 1, 2 or 4 delays the decline of intracellular free Ca2+ concentration from the peak after stimulation with ATPPC12 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and 48 h after transfection were loaded with X-rhod-1 AM and then live cells were imaged. An ECFP image was taken to allow identification of transfected and non-transfected cells and then X-rhod-1 fluorescence was monitored before stimulation and after stimulation by perfusion with 300 μM ATP. Images of ECFP and X-rhod-1 before and at the peak after stimulation are shown. After completion of the experiment, average values were collected for whole cell fluorescence for transfected and adjacent control cells. Fluorescence values were normalized to the initial fluorescence for each cell and the results are shown as means±S.E.M. The numbers of cells for each condition were as follows: KChIP1, 19 control and 17 transfected; KChIP2, 35 control and 29 transfected; KChIP3, 19 control and 21 transfected; KChIP4, 22 control and 22 transfected. * Indicates time points at which the values for KChIP3–ECFP-transfected cells were significantly different from control values based on use of a Student's t test with P values less than 0.05.
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Figure 5: Expression of KChIP3, but not KChIPs 1, 2 or 4 delays the decline of intracellular free Ca2+ concentration from the peak after stimulation with ATPPC12 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and 48 h after transfection were loaded with X-rhod-1 AM and then live cells were imaged. An ECFP image was taken to allow identification of transfected and non-transfected cells and then X-rhod-1 fluorescence was monitored before stimulation and after stimulation by perfusion with 300 μM ATP. Images of ECFP and X-rhod-1 before and at the peak after stimulation are shown. After completion of the experiment, average values were collected for whole cell fluorescence for transfected and adjacent control cells. Fluorescence values were normalized to the initial fluorescence for each cell and the results are shown as means±S.E.M. The numbers of cells for each condition were as follows: KChIP1, 19 control and 17 transfected; KChIP2, 35 control and 29 transfected; KChIP3, 19 control and 21 transfected; KChIP4, 22 control and 22 transfected. * Indicates time points at which the values for KChIP3–ECFP-transfected cells were significantly different from control values based on use of a Student's t test with P values less than 0.05.

Mentions: Overexpression of KChIP3 has been shown to result in an increase in the Ca2+ content of the ER (endoplasmic reticulum) [33] and to reduce the expression of one of the three [37] plasma membrane Na+/Ca2+ exchangers, NCX3 [32]. Therefore it is possible that the effect of KChIP3 on GH secretion could have been due to a modification in the Ca2+ signal following ATP stimulation. To find out whether this was the case, and if any effect was specific for KChIP3, PC12 cells were transfected with each of the KChIPs tagged with ECFP, the cells were loaded with the calcium indicator X-rhod-1 and changes in [Ca2+]i in response to ATP were monitored [38]. Before and after stimulation, cellular fluorescence levels were monitored from high-ECFP-expressing cells and adjacent control non-transfected cells and the data for expressing and non-transfected cells on the same coverslips were directly compared. Expression of ECFP alone had no effect on the [Ca2+]i changes during ATP stimulation (results not shown). Expression of KChIPs 1–4 had no significant effect on the peak increase in [Ca2+]i following ATP addition (Figure 5). KChIPs 1, 2 and 4 had no detectable effect on any other aspect of the Ca2+ signal but KChIP3–ECFP resulted in a significantly elevated [Ca2+]i during later times as the Ca2+ concentration was slowly declining (Figure 5C).


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)

Expression of KChIP3, but not KChIPs 1, 2 or 4 delays the decline of intracellular free Ca2+ concentration from the peak after stimulation with ATPPC12 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and 48 h after transfection were loaded with X-rhod-1 AM and then live cells were imaged. An ECFP image was taken to allow identification of transfected and non-transfected cells and then X-rhod-1 fluorescence was monitored before stimulation and after stimulation by perfusion with 300 μM ATP. Images of ECFP and X-rhod-1 before and at the peak after stimulation are shown. After completion of the experiment, average values were collected for whole cell fluorescence for transfected and adjacent control cells. Fluorescence values were normalized to the initial fluorescence for each cell and the results are shown as means±S.E.M. The numbers of cells for each condition were as follows: KChIP1, 19 control and 17 transfected; KChIP2, 35 control and 29 transfected; KChIP3, 19 control and 21 transfected; KChIP4, 22 control and 22 transfected. * Indicates time points at which the values for KChIP3–ECFP-transfected cells were significantly different from control values based on use of a Student's t test with P values less than 0.05.
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Related In: Results  -  Collection

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Figure 5: Expression of KChIP3, but not KChIPs 1, 2 or 4 delays the decline of intracellular free Ca2+ concentration from the peak after stimulation with ATPPC12 cells were transfected to express KChIPs 1–4 as ECFP-tagged constructs and 48 h after transfection were loaded with X-rhod-1 AM and then live cells were imaged. An ECFP image was taken to allow identification of transfected and non-transfected cells and then X-rhod-1 fluorescence was monitored before stimulation and after stimulation by perfusion with 300 μM ATP. Images of ECFP and X-rhod-1 before and at the peak after stimulation are shown. After completion of the experiment, average values were collected for whole cell fluorescence for transfected and adjacent control cells. Fluorescence values were normalized to the initial fluorescence for each cell and the results are shown as means±S.E.M. The numbers of cells for each condition were as follows: KChIP1, 19 control and 17 transfected; KChIP2, 35 control and 29 transfected; KChIP3, 19 control and 21 transfected; KChIP4, 22 control and 22 transfected. * Indicates time points at which the values for KChIP3–ECFP-transfected cells were significantly different from control values based on use of a Student's t test with P values less than 0.05.
Mentions: Overexpression of KChIP3 has been shown to result in an increase in the Ca2+ content of the ER (endoplasmic reticulum) [33] and to reduce the expression of one of the three [37] plasma membrane Na+/Ca2+ exchangers, NCX3 [32]. Therefore it is possible that the effect of KChIP3 on GH secretion could have been due to a modification in the Ca2+ signal following ATP stimulation. To find out whether this was the case, and if any effect was specific for KChIP3, PC12 cells were transfected with each of the KChIPs tagged with ECFP, the cells were loaded with the calcium indicator X-rhod-1 and changes in [Ca2+]i in response to ATP were monitored [38]. Before and after stimulation, cellular fluorescence levels were monitored from high-ECFP-expressing cells and adjacent control non-transfected cells and the data for expressing and non-transfected cells on the same coverslips were directly compared. Expression of ECFP alone had no effect on the [Ca2+]i changes during ATP stimulation (results not shown). Expression of KChIPs 1–4 had no significant effect on the peak increase in [Ca2+]i following ATP addition (Figure 5). KChIPs 1, 2 and 4 had no detectable effect on any other aspect of the Ca2+ signal but KChIP3–ECFP resulted in a significantly elevated [Ca2+]i during later times as the Ca2+ concentration was slowly declining (Figure 5C).

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