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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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Reconstitution of native ISA channel from CG cells by heterologous expression in oocytes.(A) Outward transient currents elicited from CG cells and oocytes expressing Kv4.2, a mixture of DPP6a and DPP6K at 1∶2 ratio, and either KChIP3a or KChIP4bL. From a holding potential of −100 mV, either a 200-ms (CG cells) or 1-sec (oocytes) step depolarizations were made from −100 mV to +40 mV at 10 mV increments. (B) Overlapped normalized current traces at +40 mV from the indicated channels. (C) Time constants of inactivation at indicated membrane potentials for ISA from CG cells, Kv4.2+KChIP3a+DPP6a+DPP6K (1∶2), and Kv4.2+KChIP4bL+DPP6a+DPP6K (1∶2). (D) Recovery from inactivation at −100 mV, measured using the two-pulse protocol. (E) Normalized peak conductance-voltage relations (Gp/Gp,max) and steady-state inactivation curves (I/Imax) for ISA from CG cells and reconstituted channel complexes.
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pone-0038205-g007: Reconstitution of native ISA channel from CG cells by heterologous expression in oocytes.(A) Outward transient currents elicited from CG cells and oocytes expressing Kv4.2, a mixture of DPP6a and DPP6K at 1∶2 ratio, and either KChIP3a or KChIP4bL. From a holding potential of −100 mV, either a 200-ms (CG cells) or 1-sec (oocytes) step depolarizations were made from −100 mV to +40 mV at 10 mV increments. (B) Overlapped normalized current traces at +40 mV from the indicated channels. (C) Time constants of inactivation at indicated membrane potentials for ISA from CG cells, Kv4.2+KChIP3a+DPP6a+DPP6K (1∶2), and Kv4.2+KChIP4bL+DPP6a+DPP6K (1∶2). (D) Recovery from inactivation at −100 mV, measured using the two-pulse protocol. (E) Normalized peak conductance-voltage relations (Gp/Gp,max) and steady-state inactivation curves (I/Imax) for ISA from CG cells and reconstituted channel complexes.

Mentions: In recordings using whole-cell patch clamp from acute cerebellar slices, we found that the native ISA of CG cells rises rapidly and decays with a clearly evident two exponential time course, in agreement with previous reports [38], [39]. As the membrane potential becomes more positive, the rapid inactivating component (τ-1≃11 ms at +40 mV) becomes more prominent than the slow inactivating phase (τ-2≃120 ms at +40 mV) (Fig. 7A). As for the voltage dependence of the two inactivating components, the fast phase of inactivation is voltage-independent above −10 mV, but for the slow phase, inactivation becomes progressively slower with increasing depolarization (Fig. 7C). This inverse relationship between voltage and inactivation kinetics has been described in native ISA of CG cells by analyzing time constants of exponential fittings or half-inactivation times [29], [39].


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)

Reconstitution of native ISA channel from CG cells by heterologous expression in oocytes.(A) Outward transient currents elicited from CG cells and oocytes expressing Kv4.2, a mixture of DPP6a and DPP6K at 1∶2 ratio, and either KChIP3a or KChIP4bL. From a holding potential of −100 mV, either a 200-ms (CG cells) or 1-sec (oocytes) step depolarizations were made from −100 mV to +40 mV at 10 mV increments. (B) Overlapped normalized current traces at +40 mV from the indicated channels. (C) Time constants of inactivation at indicated membrane potentials for ISA from CG cells, Kv4.2+KChIP3a+DPP6a+DPP6K (1∶2), and Kv4.2+KChIP4bL+DPP6a+DPP6K (1∶2). (D) Recovery from inactivation at −100 mV, measured using the two-pulse protocol. (E) Normalized peak conductance-voltage relations (Gp/Gp,max) and steady-state inactivation curves (I/Imax) for ISA from CG cells and reconstituted channel complexes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038205-g007: Reconstitution of native ISA channel from CG cells by heterologous expression in oocytes.(A) Outward transient currents elicited from CG cells and oocytes expressing Kv4.2, a mixture of DPP6a and DPP6K at 1∶2 ratio, and either KChIP3a or KChIP4bL. From a holding potential of −100 mV, either a 200-ms (CG cells) or 1-sec (oocytes) step depolarizations were made from −100 mV to +40 mV at 10 mV increments. (B) Overlapped normalized current traces at +40 mV from the indicated channels. (C) Time constants of inactivation at indicated membrane potentials for ISA from CG cells, Kv4.2+KChIP3a+DPP6a+DPP6K (1∶2), and Kv4.2+KChIP4bL+DPP6a+DPP6K (1∶2). (D) Recovery from inactivation at −100 mV, measured using the two-pulse protocol. (E) Normalized peak conductance-voltage relations (Gp/Gp,max) and steady-state inactivation curves (I/Imax) for ISA from CG cells and reconstituted channel complexes.
Mentions: In recordings using whole-cell patch clamp from acute cerebellar slices, we found that the native ISA of CG cells rises rapidly and decays with a clearly evident two exponential time course, in agreement with previous reports [38], [39]. As the membrane potential becomes more positive, the rapid inactivating component (τ-1≃11 ms at +40 mV) becomes more prominent than the slow inactivating phase (τ-2≃120 ms at +40 mV) (Fig. 7A). As for the voltage dependence of the two inactivating components, the fast phase of inactivation is voltage-independent above −10 mV, but for the slow phase, inactivation becomes progressively slower with increasing depolarization (Fig. 7C). This inverse relationship between voltage and inactivation kinetics has been described in native ISA of CG cells by analyzing time constants of exponential fittings or half-inactivation times [29], [39].

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