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Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

Jerng HH, Pfaffinger PJ - PLoS ONE (2012)

Bottom Line: Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve.When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function.A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America. hjerng@cns.bcm.edu

ABSTRACT
Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I(SA) functional properties in specific neuronal populations.

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Analysis of DPP6 splice variant expression in CG cells.A) DPP6 gene shows a conserved set of four alternative first exons producing the protein variants DPP6a, DPP6K, DPP6L and DPP6S. B) qRT-PCR using SYBR Green Fluorescence for DPP6 variants from three brain regions: cortex, cerebellum and hippocampus. DPP6a and DPP6K show enhanced expression in cerebellum. C) Normalization controls used to correct for primer amplification efficiency differences. Amplification targets from GAPDH and two DPLP variants were diluted and used to construct amplification curves. Common GAPDH signal was used to ensure consistent dilution of standards. D) Relative expression levels of DPP6 variants in cerebellum following normalization. Due to high levels of expression in CG cells and the abundance of these neurons, these signals essentially report the relative expression of DPP6 variants in CG cells.
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pone-0038205-g001: Analysis of DPP6 splice variant expression in CG cells.A) DPP6 gene shows a conserved set of four alternative first exons producing the protein variants DPP6a, DPP6K, DPP6L and DPP6S. B) qRT-PCR using SYBR Green Fluorescence for DPP6 variants from three brain regions: cortex, cerebellum and hippocampus. DPP6a and DPP6K show enhanced expression in cerebellum. C) Normalization controls used to correct for primer amplification efficiency differences. Amplification targets from GAPDH and two DPLP variants were diluted and used to construct amplification curves. Common GAPDH signal was used to ensure consistent dilution of standards. D) Relative expression levels of DPP6 variants in cerebellum following normalization. Due to high levels of expression in CG cells and the abundance of these neurons, these signals essentially report the relative expression of DPP6 variants in CG cells.

Mentions: To determine the relative expression of different DPP6 variants in CG cells, we quantified the mRNA expression levels for DPP6 variants using qRT-PCR. PCR primers were designed to amplify variant-specific DNA sequences for each of the four DPP6 N-terminal variants expressed in rat brain (Fig. 1A). Forward primers specific for the different DPP6 Exon 1's were paired with common reverse primers in Exons 2 and 3 (see Methods). Amplification curves from the cortex, cerebellum, and hippocampus showed that DPP6K and DPP6a are expressed at higher levels in cerebellum, whereas DPP6S and DPP6L are expressed at similar levels in these three brain regions (Fig. 1B). While these results showed a clear difference in the regional expression patterns for different DPP6 variants, they could not be directly compared across variants without precisely knowing the efficiencies of the different primer sets.


Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

Jerng HH, Pfaffinger PJ - PLoS ONE (2012)

Analysis of DPP6 splice variant expression in CG cells.A) DPP6 gene shows a conserved set of four alternative first exons producing the protein variants DPP6a, DPP6K, DPP6L and DPP6S. B) qRT-PCR using SYBR Green Fluorescence for DPP6 variants from three brain regions: cortex, cerebellum and hippocampus. DPP6a and DPP6K show enhanced expression in cerebellum. C) Normalization controls used to correct for primer amplification efficiency differences. Amplification targets from GAPDH and two DPLP variants were diluted and used to construct amplification curves. Common GAPDH signal was used to ensure consistent dilution of standards. D) Relative expression levels of DPP6 variants in cerebellum following normalization. Due to high levels of expression in CG cells and the abundance of these neurons, these signals essentially report the relative expression of DPP6 variants in CG cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366920&req=5

pone-0038205-g001: Analysis of DPP6 splice variant expression in CG cells.A) DPP6 gene shows a conserved set of four alternative first exons producing the protein variants DPP6a, DPP6K, DPP6L and DPP6S. B) qRT-PCR using SYBR Green Fluorescence for DPP6 variants from three brain regions: cortex, cerebellum and hippocampus. DPP6a and DPP6K show enhanced expression in cerebellum. C) Normalization controls used to correct for primer amplification efficiency differences. Amplification targets from GAPDH and two DPLP variants were diluted and used to construct amplification curves. Common GAPDH signal was used to ensure consistent dilution of standards. D) Relative expression levels of DPP6 variants in cerebellum following normalization. Due to high levels of expression in CG cells and the abundance of these neurons, these signals essentially report the relative expression of DPP6 variants in CG cells.
Mentions: To determine the relative expression of different DPP6 variants in CG cells, we quantified the mRNA expression levels for DPP6 variants using qRT-PCR. PCR primers were designed to amplify variant-specific DNA sequences for each of the four DPP6 N-terminal variants expressed in rat brain (Fig. 1A). Forward primers specific for the different DPP6 Exon 1's were paired with common reverse primers in Exons 2 and 3 (see Methods). Amplification curves from the cortex, cerebellum, and hippocampus showed that DPP6K and DPP6a are expressed at higher levels in cerebellum, whereas DPP6S and DPP6L are expressed at similar levels in these three brain regions (Fig. 1B). While these results showed a clear difference in the regional expression patterns for different DPP6 variants, they could not be directly compared across variants without precisely knowing the efficiencies of the different primer sets.

Bottom Line: Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve.When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function.A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Baylor College of Medicine, Houston, Texas, United States of America. hjerng@cns.bcm.edu

ABSTRACT
Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I(SA) functional properties in specific neuronal populations.

Show MeSH
Related in: MedlinePlus