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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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Co-assembly of DPP6K and DPP6a in heteromultimeric channel complexes.(A) Outward currents expressed by oocytes co-injected by various combinations of cRNAs, as elicited by depolarization to +40 mV from holding potential of −100 mV. (B) Expected rise and decay of currents if DPP6a and DPP6K subunits do not co-assemble and produce segregated channel populations containing either one alone. (C) Slowing of the time constant of fast inactivation when DPP6a mRNA changes from 100% to 10% mixed with DPP6K mRNA. To get the average value for fast inactivation, the slow phase of inactivation and non-inactivating current were described by exponential fitting and subtracted from the total current. The remaining average fast inactivation time constant was measured by taking the peak current for the fast inactivating fraction divided by its area. The average time constant measured by this method was very similar to the time constant measured by the best single exponential fit to the fast inactivating component. The black and gray lines show the predicted maximal slowing of fast inactivation with four DPP6 and two DPP6 per channel, respectively, with only 1 DPP6a subunit per channel. (D) Recovery from inactivation at −100 mV after a 200 ms-long prepulse (symbols) as compared to predicted results assuming no co-assembly of DPP6a and DPP6K (dashes).
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pone-0038205-g005: Co-assembly of DPP6K and DPP6a in heteromultimeric channel complexes.(A) Outward currents expressed by oocytes co-injected by various combinations of cRNAs, as elicited by depolarization to +40 mV from holding potential of −100 mV. (B) Expected rise and decay of currents if DPP6a and DPP6K subunits do not co-assemble and produce segregated channel populations containing either one alone. (C) Slowing of the time constant of fast inactivation when DPP6a mRNA changes from 100% to 10% mixed with DPP6K mRNA. To get the average value for fast inactivation, the slow phase of inactivation and non-inactivating current were described by exponential fitting and subtracted from the total current. The remaining average fast inactivation time constant was measured by taking the peak current for the fast inactivating fraction divided by its area. The average time constant measured by this method was very similar to the time constant measured by the best single exponential fit to the fast inactivating component. The black and gray lines show the predicted maximal slowing of fast inactivation with four DPP6 and two DPP6 per channel, respectively, with only 1 DPP6a subunit per channel. (D) Recovery from inactivation at −100 mV after a 200 ms-long prepulse (symbols) as compared to predicted results assuming no co-assembly of DPP6a and DPP6K (dashes).

Mentions: Based on our qRT-PCR results, nearly 75% of all DPP6 transcripts in CG cells consist of either DPP6a or DPP6K, both of which separately confer distinct functional properties. An important question is: What are the functional consequences of forming channels with both DPP6a and DPPK in the same channel complex? Previous study has suggested that four DPP6 proteins may be co-assemble onto a Kv4-based channel, although DPP6 in solution tends to form dimers [30], [32]. Because of variant co-assembly, there are a large number of potential DPP6 subunit compositions that can exist on native ISA channels. To begin to address these questions, DPP6a and DPP6K were co-expressed with Kv4.2 and KChIP3a at a series of different cRNA ratios (DPP6a∶DPP6K from 1∶1, 1∶2, and 1∶3). We first verified that the expression results are consistent with co-assembly of DPP6 variants on the channel. As Fig. 5A shows, even in channels expressed with a DPP6a∶DPP6K ratio of 1∶3, the rapid N-type inactivation produced by the DPP6a N-terminus is clearly evident in the current decay. This dominant effect of DPP6a would not occur if the DPP6 variants were not co-assembling and segregating onto different channels, resulting in channels that predominantly behave like DPP6K type channels (Fig. 5B). Although the DPP6a-mediated N-type inactivation is predominating, the kinetics for inactivation is clearly slowing as the fraction of DPP6K in the expression mix is increasing. As Table 2 shows, the time constant of fast component significantly increased from 6.0±0.4 ms (n = 8) for DPP6a-only channels to 8.5±0.1 ms (n = 5) for DPP6a∶DPP6K ratios of 1∶3 (P = 0.0003).


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)

Co-assembly of DPP6K and DPP6a in heteromultimeric channel complexes.(A) Outward currents expressed by oocytes co-injected by various combinations of cRNAs, as elicited by depolarization to +40 mV from holding potential of −100 mV. (B) Expected rise and decay of currents if DPP6a and DPP6K subunits do not co-assemble and produce segregated channel populations containing either one alone. (C) Slowing of the time constant of fast inactivation when DPP6a mRNA changes from 100% to 10% mixed with DPP6K mRNA. To get the average value for fast inactivation, the slow phase of inactivation and non-inactivating current were described by exponential fitting and subtracted from the total current. The remaining average fast inactivation time constant was measured by taking the peak current for the fast inactivating fraction divided by its area. The average time constant measured by this method was very similar to the time constant measured by the best single exponential fit to the fast inactivating component. The black and gray lines show the predicted maximal slowing of fast inactivation with four DPP6 and two DPP6 per channel, respectively, with only 1 DPP6a subunit per channel. (D) Recovery from inactivation at −100 mV after a 200 ms-long prepulse (symbols) as compared to predicted results assuming no co-assembly of DPP6a and DPP6K (dashes).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366920&req=5

pone-0038205-g005: Co-assembly of DPP6K and DPP6a in heteromultimeric channel complexes.(A) Outward currents expressed by oocytes co-injected by various combinations of cRNAs, as elicited by depolarization to +40 mV from holding potential of −100 mV. (B) Expected rise and decay of currents if DPP6a and DPP6K subunits do not co-assemble and produce segregated channel populations containing either one alone. (C) Slowing of the time constant of fast inactivation when DPP6a mRNA changes from 100% to 10% mixed with DPP6K mRNA. To get the average value for fast inactivation, the slow phase of inactivation and non-inactivating current were described by exponential fitting and subtracted from the total current. The remaining average fast inactivation time constant was measured by taking the peak current for the fast inactivating fraction divided by its area. The average time constant measured by this method was very similar to the time constant measured by the best single exponential fit to the fast inactivating component. The black and gray lines show the predicted maximal slowing of fast inactivation with four DPP6 and two DPP6 per channel, respectively, with only 1 DPP6a subunit per channel. (D) Recovery from inactivation at −100 mV after a 200 ms-long prepulse (symbols) as compared to predicted results assuming no co-assembly of DPP6a and DPP6K (dashes).
Mentions: Based on our qRT-PCR results, nearly 75% of all DPP6 transcripts in CG cells consist of either DPP6a or DPP6K, both of which separately confer distinct functional properties. An important question is: What are the functional consequences of forming channels with both DPP6a and DPPK in the same channel complex? Previous study has suggested that four DPP6 proteins may be co-assemble onto a Kv4-based channel, although DPP6 in solution tends to form dimers [30], [32]. Because of variant co-assembly, there are a large number of potential DPP6 subunit compositions that can exist on native ISA channels. To begin to address these questions, DPP6a and DPP6K were co-expressed with Kv4.2 and KChIP3a at a series of different cRNA ratios (DPP6a∶DPP6K from 1∶1, 1∶2, and 1∶3). We first verified that the expression results are consistent with co-assembly of DPP6 variants on the channel. As Fig. 5A shows, even in channels expressed with a DPP6a∶DPP6K ratio of 1∶3, the rapid N-type inactivation produced by the DPP6a N-terminus is clearly evident in the current decay. This dominant effect of DPP6a would not occur if the DPP6 variants were not co-assembling and segregating onto different channels, resulting in channels that predominantly behave like DPP6K type channels (Fig. 5B). Although the DPP6a-mediated N-type inactivation is predominating, the kinetics for inactivation is clearly slowing as the fraction of DPP6K in the expression mix is increasing. As Table 2 shows, the time constant of fast component significantly increased from 6.0±0.4 ms (n = 8) for DPP6a-only channels to 8.5±0.1 ms (n = 5) for DPP6a∶DPP6K ratios of 1∶3 (P = 0.0003).

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