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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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DPP6K markedly represses the recovery-accelerating effects of KChIP3a.Representative current traces generated during the two-pulse protocol used to measure recovery from inactivation for Kv4.2+KChIP3a (A), Kv4.2+KChIP3a+DPP6a (B), Kv4.2+KChIP3a+DPP6S (C), and Kv4.2+KChIP3a+DPP6K (D). A 1-sec depolarization to +50 mV was delivered to maximally inactivate the channels, followed by an increasing recovery interval at −100 mV before applying a 250-ms test pulse at +50 mV to check the degree of recovery from inactivation. (E) Fractional recovery was plotted as a function of the interval duration at −100 mV. The residual value at the end the first pulse was subtracted from the peak current values of the first and second pulses, and the fractional recovery was determined by dividing the peak value of the second pulse by that of the first pulse.
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pone-0038205-g003: DPP6K markedly represses the recovery-accelerating effects of KChIP3a.Representative current traces generated during the two-pulse protocol used to measure recovery from inactivation for Kv4.2+KChIP3a (A), Kv4.2+KChIP3a+DPP6a (B), Kv4.2+KChIP3a+DPP6S (C), and Kv4.2+KChIP3a+DPP6K (D). A 1-sec depolarization to +50 mV was delivered to maximally inactivate the channels, followed by an increasing recovery interval at −100 mV before applying a 250-ms test pulse at +50 mV to check the degree of recovery from inactivation. (E) Fractional recovery was plotted as a function of the interval duration at −100 mV. The residual value at the end the first pulse was subtracted from the peak current values of the first and second pulses, and the fractional recovery was determined by dividing the peak value of the second pulse by that of the first pulse.

Mentions: The stabilization of inactivation in DPP6K channels could be due to favoring entry into the inactivated state or slower recovery from inactivation, or both. To measure the effects of DPP6K on recovery from inactivation, we examined recovery from inactivation at −100 mV using a two-pulse protocol and plotted the fractional recovery as a function of the interpulse duration (Fig. 3A–3E). The length of the first pulse was set at 1 second to allow maximum amount of inactivation before testing for channel recovery. As shown in Figure 3A–3D, the addition of DPP6S or DPP6a dramatically accelerated the recovery kinetics of Kv4.2+KChIP3a channels. However, when DPP6K is co-expressed with Kv4.2+KChIP3a channels, the opposite effect occurs as the kinetics of recovery from inactivation are dramatically slowed (compare Fig. 3D with Fig. 3A; Fig. 3E). Furthermore, the time course requires the sum of two exponential functions for a proper description (Table 1). These results clearly show that, in the context of the native-like ternary complex channel, the highly-expressed DPP6K variant produces distinct modulation of channel function. Furthermore, they suggest that the DPP6K-specific effects are derived from its variable N-terminus.


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)

DPP6K markedly represses the recovery-accelerating effects of KChIP3a.Representative current traces generated during the two-pulse protocol used to measure recovery from inactivation for Kv4.2+KChIP3a (A), Kv4.2+KChIP3a+DPP6a (B), Kv4.2+KChIP3a+DPP6S (C), and Kv4.2+KChIP3a+DPP6K (D). A 1-sec depolarization to +50 mV was delivered to maximally inactivate the channels, followed by an increasing recovery interval at −100 mV before applying a 250-ms test pulse at +50 mV to check the degree of recovery from inactivation. (E) Fractional recovery was plotted as a function of the interval duration at −100 mV. The residual value at the end the first pulse was subtracted from the peak current values of the first and second pulses, and the fractional recovery was determined by dividing the peak value of the second pulse by that of the first pulse.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038205-g003: DPP6K markedly represses the recovery-accelerating effects of KChIP3a.Representative current traces generated during the two-pulse protocol used to measure recovery from inactivation for Kv4.2+KChIP3a (A), Kv4.2+KChIP3a+DPP6a (B), Kv4.2+KChIP3a+DPP6S (C), and Kv4.2+KChIP3a+DPP6K (D). A 1-sec depolarization to +50 mV was delivered to maximally inactivate the channels, followed by an increasing recovery interval at −100 mV before applying a 250-ms test pulse at +50 mV to check the degree of recovery from inactivation. (E) Fractional recovery was plotted as a function of the interval duration at −100 mV. The residual value at the end the first pulse was subtracted from the peak current values of the first and second pulses, and the fractional recovery was determined by dividing the peak value of the second pulse by that of the first pulse.
Mentions: The stabilization of inactivation in DPP6K channels could be due to favoring entry into the inactivated state or slower recovery from inactivation, or both. To measure the effects of DPP6K on recovery from inactivation, we examined recovery from inactivation at −100 mV using a two-pulse protocol and plotted the fractional recovery as a function of the interpulse duration (Fig. 3A–3E). The length of the first pulse was set at 1 second to allow maximum amount of inactivation before testing for channel recovery. As shown in Figure 3A–3D, the addition of DPP6S or DPP6a dramatically accelerated the recovery kinetics of Kv4.2+KChIP3a channels. However, when DPP6K is co-expressed with Kv4.2+KChIP3a channels, the opposite effect occurs as the kinetics of recovery from inactivation are dramatically slowed (compare Fig. 3D with Fig. 3A; Fig. 3E). Furthermore, the time course requires the sum of two exponential functions for a proper description (Table 1). These results clearly show that, in the context of the native-like ternary complex channel, the highly-expressed DPP6K variant produces distinct modulation of channel function. Furthermore, they suggest that the DPP6K-specific effects are derived from its variable N-terminus.

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