DPP10 splice variants are localized in distinct neuronal populations and act to differentially regulate the inactivation properties of Kv4-based ion channels

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Abstract

Dipeptidyl peptidase-like proteins (DPLs) and Kv-channel-interacting proteins (KChIPs) join Kv4 pore-forming subunits to form multi-protein complexes that underlie subthreshold A-type currents (ISA) in neuronal somatodendritic compartments. Here, we characterize the functional effects and brain distributions of N-terminal variants belonging to the DPL dipeptidyl peptidase 10 (DPP10). In the Kv4.2 + KChIP3 + DPP10 channel complex, all DPP10 variants accelerate channel gating kinetics; however, the splice variant DPP10a produces uniquely fast inactivation kinetics that accelerates with increasing depolarization. This DPP10a-specific inactivation dominates in co-expression studies with KChIP4a and other DPP10 isoforms. Real-time qRT-PCR and in situ hybridization analyses reveal differential expression of DPP10 variants in rat brain. DPP10a transcripts are prominently expressed in the cortex, whereas DPP10c and DPP10d mRNAs exhibit more diffuse distributions. Our results suggest that DPP10a underlies rapid inactivation of cortical ISA, and the regulation of isoform expression may contribute to the variable inactivation properties of ISA across different brain regions.

Introduction

Subthreshold transient K+ current (ISA) influences neuronal excitability and firing properties. The loss or reduction of ISA may contribute to increased excitability and susceptibility to induction and expression of epileptiform activity (Castro et al., 2001, Bernard et al., 2004). Localized to the somatodendritic compartments, ISA activity is also responsible for coordinating synaptic signal integration, controlling back-propagating action potentials and regulating long-term potentiation (Hoffman et al., 1997, Schoppa and Westbrook, 1999, Johnston et al., 2000, Watanabe et al., 2002).

The physiological function of ISA depends on its unique kinetic properties, including rapid inactivation, rapid activation and inactivation in the subthreshold range of membrane potentials and rapid recovery from inactivation at hyperpolarized potentials. These properties can vary significantly between ISA of different neuronal populations (reviewed by Jerng et al., 2004b). For example, at + 50 mV ISA of hippocampal CA1 and CA3 pyramidal neurons inactivate with a time constant of 26–34 ms, whereas ISA of cortical pyramidal neurons inactivate with a time constant of 7–8 ms (Hoffman et al., 1997, Martina et al., 1998, Lien et al., 2002, Klee et al., 1995, Banks et al., 1996, Bekkers, 2000, Korngreen and Sakmann, 2000). Moreover, in hippocampal CA1 and CA3 pyramidal neurons but not in cortical pyramidal neurons, ISA displays an unusual voltage dependence where the inactivation kinetics slow with increasing depolarization. Possible explanations for the variability of ISA properties include differences in (1) protein composition of the ISA channel, (2) enzymatic modifications of the proteins involved and (3) intracellular environment.

The majority of neuronal ISA is mediated by the Kv4 subfamily of voltage-dependent K+ channels (Johns et al., 1997, Shibata et al., 2000, Malin and Nerbonne, 2000). Recent studies in heterologous expression systems have suggested that the combined effects of Kv4 modulatory β-subunits, K channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like (DPL) proteins are required to produce functional A-type currents from Kv4 ternary channel complexes that resemble native neuronal ISA (Nadal et al., 2003, Jerng et al., 2005). DPLs include the genetically related dipeptidyl peptidase 6 (DPP6; also known as BSPL, DPL1 and DPPX) and dipeptidyl peptidase 10 (DPP10; also known as KIAA1492, DPRP3, DPL2 and DPPY) (Wada et al., 1992, de Lecea et al., 1994, Nagase et al., 2000, Chen et al., 2003, Qi et al., 2003, Nadal et al., 2003, Jerng et al., 2004a, Zagha et al., 2005, Ren et al., 2005). When expressed alone, Kv4 channels are transported poorly to the cell surface and express kinetic properties very different from those of native ISA channels (Shibata et al., 2003; reviewed by Jerng et al., 2004b). Co-expression with KChIPs allows Kv4 channels to reach the cell surface efficiently as well as reconstituting some of the kinetic properties of native ISA, such as rapid recovery from inactivation; however, the channels still behave differently from native ISA channels (An et al., 2000, Nadal et al., 2001). Only by the creation of ternary complexes between Kv4s, KChIPs and DPLs can other characteristic ISA functional properties such as rapid activation and rapid inactivation be reconstituted (Nadal et al., 2003, Jerng et al., 2005). The physiological significances of these observations are supported by gene knockout, dominant negative, RNA interference and co-immunoprecipitation studies that suggest that these three proteins are all part of the native channel complex responsible for producing ISA (Malin and Nerbonne, 2000, Nadal et al., 2003, Jerng et al., 2005, Hu et al., 2006, Lauver et al., 2006).

Although it is now clear that the Kv4, KChIP and DPL subunits are all important components of native ISA channels, the mechanisms responsible for producing functional diversity in these channels between different brain regions are not known. To address this question, we examined the interplay between different KChIP and DPL in generating A-type currents with unique functional properties. We characterized the distribution of a number of alternative splice isoforms of DPP10 that are expressed in rat brain. Functional reconstitution studies in heterologous expression systems showed that the inactivation properties of reconstituted ISA channels depend upon the DPP10 isoform that is expressed. In summary, our studies suggest that differential inactivation properties produced by DPP10 alternative splice variants are an important determinant of the native ISA current functional variations between different neuronal populations. Some of the results presented here have appeared in abstract form (Jerng and Pfaffinger, 2006).

Section snippets

Identification of DPP10 exon 1 variants

EST sequence databases and an analysis of DPP10 transcripts in a study on asthma suggest that DPP10 transcripts may express different 1st exons due to initiation from a variety of start sites within the DPP10 gene (Allen et al., 2003, Ren et al., 2005, Takimoto et al., 2006). To determine the variety of DPP10 transcripts made in rat brain, we performed rapid amplification of 5′ complementary DNA ends (5′ RACE) on mRNA isolated from rat brain and cloned the PCR products for DNA sequencing.

DPP10 N-terminal variants are differentially expressed in the brain and produce different functional effects

5′ RACE analysis performed on mRNAs extracted from whole rat brain as well as rat cerebellum clearly establishes the existence of three DPP10 splicing variants: DPP10a, DPP10c and DPP10d. These variants are generated by alternative splicing of the first DPP10 exon (exon 1) and differ only by their cytoplasmic N-terminal domain. The N-terminal variants of DPP10 are expressed with distinct patterns in rat brain: DPP10a expression is largely restricted to Layer 2/3 and Layer 5 of the cerebral

Molecular biology

Plasmids containing rat Kv4.2, human KChIP3, human DPP10a and human DPP6-S cDNAs were obtained as described previously (Jerng et al., 2004a, Jerng et al., 2005). Kv4.2/ΔN2-40 plasmid was constructed by subcloning a PCR fragment encoding Kv4.2 starting methionine, residues 41–630 and stop codon into pBluescript. KChIP4a cDNA was generated by purchasing a human hippocampal KChIP4b clone (IMAGE #4817099) from American Type Culture Collection (ATCC, Manassas, VA, USA) and substituting in the 5′

Acknowledgments

We thank Dr. Lily Jan for providing the Kv4.2 cDNA and Drs. Ramiro Salas and Mariella DeBiasi for their invaluable assistance and advice on the in situ hybridization experiments. We also thank Dr. Covarrubias for critical reading of the manuscript and Alison Prince for assisting in harvesting quality oocytes for the functional studies. This work was supported by a grant from the National Institute of Health (P01 NS37444).

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