A novel DPP6 isoform (DPP6-E) can account for differences between neuronal and reconstituted A-type K(+) channels

Abstract

The channels mediating most of the somatodendritic A-type K(+) current in neurons are thought to be ternary complexes of Kv4 pore-forming subunits and two types of auxiliary subunits, the K(+) channel interacting proteins (KChIPs) and dipeptidyl-peptidase-like (DPPL) proteins. The channels expressed in heterologous expression systems by mixtures of Kv4.2, KChIP1 and DPP6-S resemble in many properties the A-type current in hippocampal CA1 pyramidal neurons and cerebellar granule cells, neurons with prominent A-type K(+) currents. However, the native currents have faster kinetics. Moreover, the A-type currents in neurons in intermediary layers of the superior colliculus have even faster inactivating rates. We have characterized a new DPP6 spliced isoform, DPP6-E, that produces in heterologous cells ternary Kv4 channels with very fast kinetics. DPP6-E is selectively expressed in a few neuronal populations in brain including cerebellar granule neurons, hippocampal pyramidal cells and neurons in intermediary layers of the superior colliculus. The effects of DPP6-E explain past discrepancies between reconstituted and native Kv4 channels in some neurons, and contributes to the diversity of A-type K(+) currents in neurons.

Fig. 1

DPP6-E modulates the kinetics and voltage-dependence of Kv4 Channels expressed in HEK 293 cells. (A) Comparison of the divergent N-termini of DPP6 spliced isoforms. Common juxtamembrane and transmembrane domains encoded by a common second exon are highlighted in blue and red, respectively. Only the first 6 amino acids of the transmembrane domain are shown. Letters in red indicate conserved amino acids in the N-termini, which are encoded in alternatively spliced exons. Also included for comparison is the N-terminus of DPP10-A. Shown is the alignment of the following rodent sequences: NP 034205 (DPP6-E); NP 001012205 (DPP10-A) M76427 (DPP6-S); M76426 (DPP6-L); CF743412 (DPP6-K); and CB585942 (DPP6-D). (B) Kv4.2-mediated A-type K⁺ currents in representative HEK 293 cells transfected with Kv4.2 DNA alone or (C) Kv4.2 plus DPP6-E cDNA at a ~1:1 molar ratio obtained by whole cell recording. Cells were voltage clamped at a holding potential of −90 mV followed by depolarizing test pulses from −90 to 40 mV in 10 mV increments. (D) Normalized conductance–voltage relation (G/Gmax) and steady-state inactivation curves (I/I_max) for the I_SA in cells expressing Kv4.2 alone (black symbols) and Kv4.2 + DPP6-E (red symbols). Shown are averages from 6 cells. (E) Time course of the recovery from inactivation of the I_SA in cells transfected with Kv4.2 (black squares) and Kv4.2 plus DPP6-E (red circles) at a ~1:1 molar ratio. The currents were recorded during test pulses to +40 mV following a pulse to the same voltage separated by increasing time intervals at −110 mV.(F) Representative currents from HEK 293 cells transected with Kv4.2 or (G) Kv4.2 plus DPP6-E at a ~1:1 molar ratio recorded under conditions described in (E). Error bars represent S.E.M. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)

Fig. 2

I_SAs inactivate faster in the presence of DPP6-E than in the presence of DPP6-S. (A)–(F) Kv4.2-mediated A-type K+ currents in representative HEK 293 cells transfected with the indicated cDNAs at equimolar ratios. Cells were voltage clamped at a holding potential of −90 mV followed by depolarizing test pulses from −90 to 70 mV in 10 mV increments. (G)–(H) Plots of the time at which half of the peak current is inactivated (t1/2) against membrane potential from the currents recorded in HEK 293 cells expressing Kv4.2 with or without KChIP in the absence or presence of DPP6-E or DPP6-S as indicated. In (H) open squares show the t1/2 of inactivation of FS and late-spiking cells in the superior colliculus reported by Saito and Isa [31]. Note the similarity between these native currents and those produced by the Kv4.2, KChIP, DPP6-E complex. Error bars represent S.E.M.

Fig. 3

Distribution of DPP6-E mRNA transcripts in mouse brain. DPP6-E exon-specific cRNA probes were used to localize DPP6-E expression in mouse brain using non-radioactive in situ hybridization. Enlarged sections of specific staining including the hippocampus (A); intermediary layer of the superior colliculus (B); and cerebellum (C) are taken from the areas indicated by boxes in the full sagital section (D). Further enlargement of the staining pattern from these fields is shown in inset images. Calibration bar shows 1 mm for image in (D).

Fig. 4

Intermediary inactivation kinetics produced by Kv4–KChIP channels expressed with different ratios of DPP6-E and DPP6-S. Plotted are the t1/2 of inactivation against membrane potential for HEK 293 cells transfected with Kv4.2 and KChIP with either DPP6-S, DPP6-E or the indicated mixtures of both isoforms. Cells were voltage clamped at a holding potential of −90 mV followed by depolarizing test pulses from −20 to 40 mV in 10 mV increments. The open squares represent the t1/2 of inactivation of A-type currents recorded from cerebellar granule neurons (CGN) reported by Amarillo et al. (2008). Note that the ratio of DPP6-S to DPP6-E determines the effect of depolarization on the t1/2 of inactivation for the currents in the heterologous system and that the native currents fall within the range of kinetics produced by these mixtures. Error bars represent S.E.M.