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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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Steady-state inactivation of DPP6a∶DPP6K mixed channels.(A) Representative traces for Kv4.2+KChIP3a channels co-expressed with DPP6a alone, DPP6K alone, or with a DPP6a∶DPP6K mixture at 1∶1 or 1∶2 ratios, showing changes in steady-state inactivation at −65 mV. For the colored traces, the channels were held for 30 sec at −65 mV before pulsing to +40 mV for 250 ms to test available current. The black traces show the total currents, from test pulses where the channels were held at −100 mV and experienced no inactivation. (B) Voltage dependence of steady-state inactivation of ternary complexes with homotetrameric and heterotetrameric DPP6 subunits. (C) Progressive shifting of inactivation midpoint with increasing DPP6K ratio. The V0.5i values were plotted against the calculated DPP6K mole fraction. Model assumes independent energetic effects for each DPP6K subunit incorporated into the channel, with symbols measured V0.5i values for summed Boltzmann curves from the model.
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pone-0038205-g006: Steady-state inactivation of DPP6a∶DPP6K mixed channels.(A) Representative traces for Kv4.2+KChIP3a channels co-expressed with DPP6a alone, DPP6K alone, or with a DPP6a∶DPP6K mixture at 1∶1 or 1∶2 ratios, showing changes in steady-state inactivation at −65 mV. For the colored traces, the channels were held for 30 sec at −65 mV before pulsing to +40 mV for 250 ms to test available current. The black traces show the total currents, from test pulses where the channels were held at −100 mV and experienced no inactivation. (B) Voltage dependence of steady-state inactivation of ternary complexes with homotetrameric and heterotetrameric DPP6 subunits. (C) Progressive shifting of inactivation midpoint with increasing DPP6K ratio. The V0.5i values were plotted against the calculated DPP6K mole fraction. Model assumes independent energetic effects for each DPP6K subunit incorporated into the channel, with symbols measured V0.5i values for summed Boltzmann curves from the model.

Mentions: The most consistent impact of DPP6K is on the steady-state inactivation properties for the channel. If we compare the maximum current in response to a depolarization to +40 mV, we see that holding at −65 mV reduces the amplitude of current evoked from DPP6a containing channel in half. As the ratio of DPP6K is increased, this steady-state inactivation at −65 mV increases until only about 10% current can be evoked with DPP6K alone (Fig. 6A). The steady-state inactivation curves produced by these mixtures show the progressive leftward shift in midpoint as DPP6K expression is increased, with no discerning change in slope factor (Fig. 6B; Table 2). When DPP6a-to-DPP6K ratio reached 1∶9, the midpoint and slope factor of steady-state inactivation were not significantly different from that of channel complexes with DPP6K alone (Kv4.2+KChIP3a+DPP6a+DPP6K (1∶9): V0.5i = −73.3±0.3 mV; Si = 4.1±0.2 mV/e-fold, n = 3). If we plot the inactivation midpoint versus the mole ratio of DPP6K we see that there is a progressive shift in midpoint as the ratio of DPP6K increases. Linear regression analysis shows a strong correlation consistent with the model that DPP6 subunit incorporation produces a consistent additive energetic shift in the inactivation midpoint of −2.3 mV for each DPP6K subunit incorporated into the channel (Fig. 6C). Our results suggest that the DPP6K expression ratio may be an important parameter neurons can use to tune the location of the ISA steady-state inactivation curve relative to the neuron's resting membrane potential.


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)

Steady-state inactivation of DPP6a∶DPP6K mixed channels.(A) Representative traces for Kv4.2+KChIP3a channels co-expressed with DPP6a alone, DPP6K alone, or with a DPP6a∶DPP6K mixture at 1∶1 or 1∶2 ratios, showing changes in steady-state inactivation at −65 mV. For the colored traces, the channels were held for 30 sec at −65 mV before pulsing to +40 mV for 250 ms to test available current. The black traces show the total currents, from test pulses where the channels were held at −100 mV and experienced no inactivation. (B) Voltage dependence of steady-state inactivation of ternary complexes with homotetrameric and heterotetrameric DPP6 subunits. (C) Progressive shifting of inactivation midpoint with increasing DPP6K ratio. The V0.5i values were plotted against the calculated DPP6K mole fraction. Model assumes independent energetic effects for each DPP6K subunit incorporated into the channel, with symbols measured V0.5i values for summed Boltzmann curves from the model.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366920&req=5

pone-0038205-g006: Steady-state inactivation of DPP6a∶DPP6K mixed channels.(A) Representative traces for Kv4.2+KChIP3a channels co-expressed with DPP6a alone, DPP6K alone, or with a DPP6a∶DPP6K mixture at 1∶1 or 1∶2 ratios, showing changes in steady-state inactivation at −65 mV. For the colored traces, the channels were held for 30 sec at −65 mV before pulsing to +40 mV for 250 ms to test available current. The black traces show the total currents, from test pulses where the channels were held at −100 mV and experienced no inactivation. (B) Voltage dependence of steady-state inactivation of ternary complexes with homotetrameric and heterotetrameric DPP6 subunits. (C) Progressive shifting of inactivation midpoint with increasing DPP6K ratio. The V0.5i values were plotted against the calculated DPP6K mole fraction. Model assumes independent energetic effects for each DPP6K subunit incorporated into the channel, with symbols measured V0.5i values for summed Boltzmann curves from the model.
Mentions: The most consistent impact of DPP6K is on the steady-state inactivation properties for the channel. If we compare the maximum current in response to a depolarization to +40 mV, we see that holding at −65 mV reduces the amplitude of current evoked from DPP6a containing channel in half. As the ratio of DPP6K is increased, this steady-state inactivation at −65 mV increases until only about 10% current can be evoked with DPP6K alone (Fig. 6A). The steady-state inactivation curves produced by these mixtures show the progressive leftward shift in midpoint as DPP6K expression is increased, with no discerning change in slope factor (Fig. 6B; Table 2). When DPP6a-to-DPP6K ratio reached 1∶9, the midpoint and slope factor of steady-state inactivation were not significantly different from that of channel complexes with DPP6K alone (Kv4.2+KChIP3a+DPP6a+DPP6K (1∶9): V0.5i = −73.3±0.3 mV; Si = 4.1±0.2 mV/e-fold, n = 3). If we plot the inactivation midpoint versus the mole ratio of DPP6K we see that there is a progressive shift in midpoint as the ratio of DPP6K increases. Linear regression analysis shows a strong correlation consistent with the model that DPP6 subunit incorporation produces a consistent additive energetic shift in the inactivation midpoint of −2.3 mV for each DPP6K subunit incorporated into the channel (Fig. 6C). Our results suggest that the DPP6K expression ratio may be an important parameter neurons can use to tune the location of the ISA steady-state inactivation curve relative to the neuron's resting membrane potential.

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