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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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The DPP6K variable N-terminus is responsible for its distinct functional effects.(A) Voltage dependence of steady-state inactivation of ternary complex containing DPP6K and DPP6K/ΔN16 deletion mutant. The protocol was the same as that of Fig. 2B. (B) The kinetics of recovery from inactivation at −100 mV. The protocol was the same as that of Fig. 3. (C) Alignment of amino acid sequences of DPP6K variable N-terminus from various organisms. Consensus residues are shown in black; conservative substitutions, in gray; non-conservative substitutions, in red. Major evolutionary branch points indicated along the alignment relative to a terminus in placental mammals.
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pone-0038205-g004: The DPP6K variable N-terminus is responsible for its distinct functional effects.(A) Voltage dependence of steady-state inactivation of ternary complex containing DPP6K and DPP6K/ΔN16 deletion mutant. The protocol was the same as that of Fig. 2B. (B) The kinetics of recovery from inactivation at −100 mV. The protocol was the same as that of Fig. 3. (C) Alignment of amino acid sequences of DPP6K variable N-terminus from various organisms. Consensus residues are shown in black; conservative substitutions, in gray; non-conservative substitutions, in red. Major evolutionary branch points indicated along the alignment relative to a terminus in placental mammals.

Mentions: To determine whether it is the presence of the DPP6K N-terminus rather than the absence of DPP6a or DPP6S N-termini that is responsible for the DPP6K-differential effects, we deleted the DPP6K Exon 1K (E1K, Fig. 1A) sequence and generated the DPP6K mutant DPP6K/ΔN16 which initiates off of the native Met-17. Measurement of the steady-state inactivation of Kv4.2+KChIP3a channels co-expressed with wild-type DPP6K or with the DPP6K/ΔN16 deletion mutant showed that removing the unique N-terminus eliminates the DPP6K specific effects (Fig. 4A; Table 1). DPP6K/ΔN16 exhibits a large depolarization in the voltage of half-inactivation compared to DPP6K, with a final value similar to that of Kv4.2+KChIP3a+DPP6S channels. A parallel regulation of recovery from inactivation was also detected where slow recovery is lost when the variable N-terminus is deleted from DPP6K, resulting in recovery that is just as fast as ternary complex channels expressing DPP6S (Fig. 4B; Table 1). In conclusion, the highly conserved N-terminal domain of DPP6K is responsible for producing the unique functional effects of this specific variant.


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)

The DPP6K variable N-terminus is responsible for its distinct functional effects.(A) Voltage dependence of steady-state inactivation of ternary complex containing DPP6K and DPP6K/ΔN16 deletion mutant. The protocol was the same as that of Fig. 2B. (B) The kinetics of recovery from inactivation at −100 mV. The protocol was the same as that of Fig. 3. (C) Alignment of amino acid sequences of DPP6K variable N-terminus from various organisms. Consensus residues are shown in black; conservative substitutions, in gray; non-conservative substitutions, in red. Major evolutionary branch points indicated along the alignment relative to a terminus in placental mammals.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038205-g004: The DPP6K variable N-terminus is responsible for its distinct functional effects.(A) Voltage dependence of steady-state inactivation of ternary complex containing DPP6K and DPP6K/ΔN16 deletion mutant. The protocol was the same as that of Fig. 2B. (B) The kinetics of recovery from inactivation at −100 mV. The protocol was the same as that of Fig. 3. (C) Alignment of amino acid sequences of DPP6K variable N-terminus from various organisms. Consensus residues are shown in black; conservative substitutions, in gray; non-conservative substitutions, in red. Major evolutionary branch points indicated along the alignment relative to a terminus in placental mammals.
Mentions: To determine whether it is the presence of the DPP6K N-terminus rather than the absence of DPP6a or DPP6S N-termini that is responsible for the DPP6K-differential effects, we deleted the DPP6K Exon 1K (E1K, Fig. 1A) sequence and generated the DPP6K mutant DPP6K/ΔN16 which initiates off of the native Met-17. Measurement of the steady-state inactivation of Kv4.2+KChIP3a channels co-expressed with wild-type DPP6K or with the DPP6K/ΔN16 deletion mutant showed that removing the unique N-terminus eliminates the DPP6K specific effects (Fig. 4A; Table 1). DPP6K/ΔN16 exhibits a large depolarization in the voltage of half-inactivation compared to DPP6K, with a final value similar to that of Kv4.2+KChIP3a+DPP6S channels. A parallel regulation of recovery from inactivation was also detected where slow recovery is lost when the variable N-terminus is deleted from DPP6K, resulting in recovery that is just as fast as ternary complex channels expressing DPP6S (Fig. 4B; Table 1). In conclusion, the highly conserved N-terminal domain of DPP6K is responsible for producing the unique functional effects of this specific variant.

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