Limits...
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.

Show MeSH

Related in: MedlinePlus

DPP6K dramatically accelerates inactivation kinetics and leftward shifts steady-state inactivation curve.Xenopus oocytes were injected with cRNAs encoding Kv4.2 and KChIP3a along with either DPP6a, DPP6S, or DPP6K. Transient currents were recorded using two-electrode voltage clamp. (A) Representative normalized current traces generated by voltage steps to +50 mV from a holding potential of −100 mV for 1 sec. Only the first 500 ms are shown. (B) Voltage dependence of steady-state inactivation for the various channel complexes. The steady-state inactivation protocol consisted of a 10-sec prepulse at the indicated potentials and a 250-ms test pulse to +50 mV, with an inter-episode interval of 5 secs. The fraction of available current (I/Imax) was plotted against the prepulse membrane potential. Data are shown as mean ± SEM, and the lines represent fits using Boltzmann functions.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366920&req=5

pone-0038205-g002: DPP6K dramatically accelerates inactivation kinetics and leftward shifts steady-state inactivation curve.Xenopus oocytes were injected with cRNAs encoding Kv4.2 and KChIP3a along with either DPP6a, DPP6S, or DPP6K. Transient currents were recorded using two-electrode voltage clamp. (A) Representative normalized current traces generated by voltage steps to +50 mV from a holding potential of −100 mV for 1 sec. Only the first 500 ms are shown. (B) Voltage dependence of steady-state inactivation for the various channel complexes. The steady-state inactivation protocol consisted of a 10-sec prepulse at the indicated potentials and a 250-ms test pulse to +50 mV, with an inter-episode interval of 5 secs. The fraction of available current (I/Imax) was plotted against the prepulse membrane potential. Data are shown as mean ± SEM, and the lines represent fits using Boltzmann functions.

Mentions: The high level of DPP6K in CG cells raised the prospect that this variant plays a special role in shaping ISA properties in CG cells. A previous study of DPP6K did not identify any significant unique functional effect when co-expressed with Kv4.2 [19]; however, since the native ISA channels also contain KChIP proteins, we sought to re-examine this question in a native-like ternary complex by combining DPP6K with Kv4.2 and KChIP3a, which are also highly expressed in CG cells [33], [34], [35], [36]. Following co-expression of DPP6K with Kv4.2 and KChIP3a in Xenopus oocytes the DPP6K-containing channels were found to have significant functional differences from channels expressed with other DPP6 variants. Similar to other DPP6 variants, activation and inactivation gating kinetics in the presence of DPP6K are accelerated compared to channels lacking DPP6; however, the extent of acceleration of inactivation is dependent on the variant being tested (Fig. 2A). To quantify the observed changes, the development of macroscopic inactivation at +40 mV in ternary complex channels containing DPP6a, DPP6S, or DPP6K were all adequately described by using the sum of two exponential terms (Table 1). Inactivation in the presence of DPP6K is not as fast as DPP6a, which has at its N-terminus an inactivation peptide that confers a distinctive ultra-fast, N-type inactivation with a τ of ≃6 ms [22]. However, DPP6K-associated channels inactivate significantly more rapidly than channels expressed with the DPP6S variant (Fig. 2A). Our analysis showed that the faster inactivation associated with DPP6K results not from changes in the time constants of the two components but rather a significant increase in the contribution from the fast inactivating component with a concomitant decrease in the contribution from the slow inactivating component (Table 1). The steady-state inactivation properties measured with DPP6K were also remarkably different from channels formed from other subunit combinations (Fig. 2B). The voltage of half-inactivation for Kv4.2+KChIP3a+DPP6K channels is consistently more hyperpolarized than Kv4.2+KChIP3a channels, Kv4.2+KChIP3a+DPP6S channels, or Kv4.2+KChIP3a+DPP6a channels (Table 1). In addition to a leftward shift in midpoint, the slope factor of steady-state inactivation for channels containing DPP6K is also significantly reduced than seen with DPP6S or DPP6a, but similar to channels lacking DPP6. These combined effects cause A-type channels containing DPP6K to favor the inactivated state at subthreshold membrane potentials compared to channels containing other DPP6 variants.


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 dramatically accelerates inactivation kinetics and leftward shifts steady-state inactivation curve.Xenopus oocytes were injected with cRNAs encoding Kv4.2 and KChIP3a along with either DPP6a, DPP6S, or DPP6K. Transient currents were recorded using two-electrode voltage clamp. (A) Representative normalized current traces generated by voltage steps to +50 mV from a holding potential of −100 mV for 1 sec. Only the first 500 ms are shown. (B) Voltage dependence of steady-state inactivation for the various channel complexes. The steady-state inactivation protocol consisted of a 10-sec prepulse at the indicated potentials and a 250-ms test pulse to +50 mV, with an inter-episode interval of 5 secs. The fraction of available current (I/Imax) was plotted against the prepulse membrane potential. Data are shown as mean ± SEM, and the lines represent fits using Boltzmann functions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038205-g002: DPP6K dramatically accelerates inactivation kinetics and leftward shifts steady-state inactivation curve.Xenopus oocytes were injected with cRNAs encoding Kv4.2 and KChIP3a along with either DPP6a, DPP6S, or DPP6K. Transient currents were recorded using two-electrode voltage clamp. (A) Representative normalized current traces generated by voltage steps to +50 mV from a holding potential of −100 mV for 1 sec. Only the first 500 ms are shown. (B) Voltage dependence of steady-state inactivation for the various channel complexes. The steady-state inactivation protocol consisted of a 10-sec prepulse at the indicated potentials and a 250-ms test pulse to +50 mV, with an inter-episode interval of 5 secs. The fraction of available current (I/Imax) was plotted against the prepulse membrane potential. Data are shown as mean ± SEM, and the lines represent fits using Boltzmann functions.
Mentions: The high level of DPP6K in CG cells raised the prospect that this variant plays a special role in shaping ISA properties in CG cells. A previous study of DPP6K did not identify any significant unique functional effect when co-expressed with Kv4.2 [19]; however, since the native ISA channels also contain KChIP proteins, we sought to re-examine this question in a native-like ternary complex by combining DPP6K with Kv4.2 and KChIP3a, which are also highly expressed in CG cells [33], [34], [35], [36]. Following co-expression of DPP6K with Kv4.2 and KChIP3a in Xenopus oocytes the DPP6K-containing channels were found to have significant functional differences from channels expressed with other DPP6 variants. Similar to other DPP6 variants, activation and inactivation gating kinetics in the presence of DPP6K are accelerated compared to channels lacking DPP6; however, the extent of acceleration of inactivation is dependent on the variant being tested (Fig. 2A). To quantify the observed changes, the development of macroscopic inactivation at +40 mV in ternary complex channels containing DPP6a, DPP6S, or DPP6K were all adequately described by using the sum of two exponential terms (Table 1). Inactivation in the presence of DPP6K is not as fast as DPP6a, which has at its N-terminus an inactivation peptide that confers a distinctive ultra-fast, N-type inactivation with a τ of ≃6 ms [22]. However, DPP6K-associated channels inactivate significantly more rapidly than channels expressed with the DPP6S variant (Fig. 2A). Our analysis showed that the faster inactivation associated with DPP6K results not from changes in the time constants of the two components but rather a significant increase in the contribution from the fast inactivating component with a concomitant decrease in the contribution from the slow inactivating component (Table 1). The steady-state inactivation properties measured with DPP6K were also remarkably different from channels formed from other subunit combinations (Fig. 2B). The voltage of half-inactivation for Kv4.2+KChIP3a+DPP6K channels is consistently more hyperpolarized than Kv4.2+KChIP3a channels, Kv4.2+KChIP3a+DPP6S channels, or Kv4.2+KChIP3a+DPP6a channels (Table 1). In addition to a leftward shift in midpoint, the slope factor of steady-state inactivation for channels containing DPP6K is also significantly reduced than seen with DPP6S or DPP6a, but similar to channels lacking DPP6. These combined effects cause A-type channels containing DPP6K to favor the inactivated state at subthreshold membrane potentials compared to channels containing other DPP6 variants.

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