Kv channel-interacting proteins (KChIPs) are auxiliary subunits of the heteromultimeric channel complexes that underlie neuronal I(SA), the subthreshold transient K(+) current that dynamically regulates membrane excitability, action potential firing properties, and long term potentiation. KChIPs form cytoplasmic associations with the principal pore-forming Kv4 subunits and typically mediate enhanced surface expression and accelerated recovery from depolarization-induced inactivation. An exception is KChIP4a, which dramatically suppresses Kv4 inactivation while promoting neither surface expression nor recovery. These unusual properties are attributed to the effects of a K channel inactivation suppressor domain (KISD) encoded within the variable N terminus of KChIP4a. Here, we have functionally and biochemically characterized two brain KChIP isoforms, KChIP2x and KChIP3x (also known as KChIP3b) and show that they also contain a functional KISD. Like KChIP4a and in contrast with non-KISD-containing KChIPs, both KChIP2x and KChIP3x strongly suppress inactivation and slow activation and inhibit the typical increases in surface expression of Kv4.2 channels. We then examined the properties of the KISD to determine potential mechanisms for its action. Subcellular fractionation shows that KChIP4a, KChIP2x, and KChIP3x are highly associated with the membrane fraction. Fluorescent confocal imaging of enhanced green fluorescent proteins (eGFP) N-terminally fused with KISD in HEK293T cells indicates that KISDs of KChIP4a, KChIP2x, and KChIP3x all autonomously target eGFP to intracellular membranes. Cell surface biotinylation experiments on KChIP4a indicate that the N terminus is exposed extracellularly, consistent with a transmembrane KISD. In summary, KChIP4a, KChIP2x, and KChIP3x comprise a novel class of KChIP isoforms characterized by an unusual transmembrane domain at their N termini that modulates Kv4 channel gating and trafficking.

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains.

A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels.

Voltage-gated potassium (Kv) channels play important roles in regulating the excitability of myocytes and neurons. Kv4.2 is the primary alpha-subunit of the channel that produces the A-type K(+) current in CA1 pyramidal neurons of the hippocampus, which is critically involved in the regulation of dendritic excitability and plasticity. K(+) channel-interacting proteins, KChIPs (KChIP1-4), associate with the N-terminal of Kv4.2 and modulate the channel's biophysical properties, turnover rate and surface expression. In the present study, we investigated the role of Kv4.2 C-terminal PKA phosphorylation site S552 in the KChIP4a-mediated effects on Kv4.2 channel trafficking. We found that while interaction between Kv4.2 and KChIP4a does not require PKA phosphorylation of Kv4.2(S552), phosphorylation of this site is necessary for both enhanced stabilization and membrane expression of Kv4.2 channel complexes produced by KChIP4a. Enhanced surface expression and protein stability conferred by co-expression of Kv4.2 with other KChIP isoforms did not require PKA phosphorylation of Kv4.2 S552. Finally, we identify A-kinase anchoring proteins (AKAPs) as Kv4.2 binding partners, allowing for discrete local PKA signaling. These data demonstrate that PKA phosphorylation of Kv4.2 plays an important role in the trafficking of Kv4.2 through its specific interaction with KChIP4a.

Voltage-gated potassium (Kv) channels are important and diverse determinants of neuronal excitability and exhibit specific expression patterns throughout the brain. Among Kv channels, Kv4 channels are major determinants of somatodendritic A-type current and are essential in controlling the amplitude of backpropagating action potentials (BAPs) into neuronal dendrites. BAPs have been well studied in a variety of neurons, and have been recently described in hippocampal and cortical interneurons, a heterogeneous population of GABAergic inhibitory cells that regulate activity of principal cells and neuronal networks. We used well-characterized mouse monoclonal antibodies against the Kv4.3 and potassium channel interacting protein (KChIP) 1 subunits of A-type Kv channels, and antibodies against different interneuron markers in single- and double-label immunohistochemistry experiments to analyze the expression patterns of Kv4.3 and KChIP1 in hippocampal Ammon's horn (CA1) neurons. Immunohistochemistry was performed on 40 mum rat brain sections using nickel-enhanced diaminobenzidine staining or multiple-label immunofluorescence. Our results show that Kv4.3 and KChIP1 component subunits of A-type channels are co-localized in the soma and dendrites of a large number of GABAergic hippocampal interneurons. These subunits co-localize extensively but not completely with markers defining the four major interneuron subpopulations tested (parvalbumin, calbindin, calretinin, and somatostatin). These results suggest that CA1 hippocampal interneurons can be divided in two groups according to the expression of Kv4.3/KChIP1 channel subunits. Antibodies against Kv4.3 and KChIP1 represent an important new tool for identifying a subpopulation of hippocampal interneurons with a unique dendritic A-type channel complement and ability to control BAPs.

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.

Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

An increasing number of observations suggest an important role for voltage-gated potassium (Kv) channels in epilepsy. We studied the cell-specific distribution of Kv4.2, phosphorylated (p) Kv4.2 and the Kv4.2 interacting protein NCS-1 using immunocytochemistry in different epilepsy-associated focal lesions. In hippocampal sclerosis (HS), Kv4.2 and pKv4.2 immunoreactivity (IR) was reduced in the neuropil in regions with prominent neuronal cell loss. In both HS and malformations of cortical development (MCD), intense labeling was found in neuronal somata, but not in dendrites. Strong NCS-1 IR was observed in neurons in all lesion types. Western blot analysis demonstrated an increase of total Kv4.2 in all lesions and activation of the ERK pathway in HS and ganglioglioma. These findings indicate that Kv4.2 is expressed in both neuronal and glial cells and its regulation may involve potassium channel interacting proteins, alterations in the subcellular localization of the channel, as well as phosphorylation-mediated posttranslational modifications.

Kv4.2 is the major voltage-gated K(+) (Kv) channel alpha subunit responsible for the somatodendritic transient or A-type current I(SA) that activates at subthreshold membrane potentials. Stable association of Kv4.2 with diverse auxiliary subunits and reversible Kv4.2 phosphorylation regulate I(SA) function. Two classes of auxiliary subunits play distinct roles in modulating the biophysical properties of Kv4.2: dipeptidyl-peptidase-like type II transmembrane proteins typified by DPPX-S, and cytoplasmic Ca(2+) binding proteins known as K(+) channel interacting proteins (KChIPs). Here, we characterize the convergent roles that DPPX-S and KChIPs play as component subunits of Kv4.2 channel complexes. We coexpressed DPPX-S with Kv4.2 in heterologous cells and found a dramatic redistribution of Kv4.2, releasing it from intracellular retention and allowing plasma membrane expression, as well as altered Kv4.2 phosphorylation, detergent solubility, and stability. These changes are remarkably similar to those obtained upon coexpression of Kv4.2 with the structurally distinct KChIPs1-3 auxiliary subunits. KChIP4a, which negatively affects the impact of other KChIPs on Kv4.2, also inhibits the effects of DPPX-S, consistent with the formation of a ternary complex of Kv4.2, DPPX-S, and KChIPs early in channel biosynthesis. Tandem MS analyses reveal that coexpression with DPPX-S or KChIP2 leads to a pattern of Kv4.2 phosphorylation in heterologous cells similar to that observed in brain, but lacking in cells expressing Kv4.2 alone. In conclusion, transmembrane DPPX-S and cytoplasmic KChIPs, despite having distinct structures and binding sites on Kv4.2, exert similar effects on Kv4.2 trafficking, but distinct effects on Kv4.2 gating.

Post-translational conjugation of Small Ubiquitin-like Modifier (SUMO) peptides to lysine (K) residues on target proteins alters their interactions. SUMOylation of a target protein can either promote its interaction with other proteins that possess SUMO binding domains, or it can prevent target protein interactions that normally occur in the absence of SUMOylation. One subclass of voltage-gated potassium channels that mediates an A-type current, IA, exists as a ternary complex comprising Kv4 pore-forming subunits, Kv channel interacting proteins (KChIP) and transmembrane dipeptidyl peptidase like proteins (DPPL). SUMOylation could potentially regulate intra- and/or intermolecular interactions within the complex. This study began to test this hypothesis and showed that Kv4.2 channels were SUMOylated in the rat brain and in human embryonic kidney (HEK) cells expressing a GFP-tagged mouse Kv4.2 channel (Kv4.2g). Prediction software identified two putative SUMOylation sites in the Kv4.2 C-terminus at K437 and K579. These sites were conserved across mouse, rat, and human Kv4.2 channels and across mouse Kv4 isoforms. Increasing Kv4.2g SUMOylation at each site by ~30% produced a significant ~22%-50% decrease in IA Gmax, and a ~70%-95% increase in channel surface expression. Site-directed mutagenesis of Kv4.2g showed that K437 SUMOylation regulated channel surface expression, while K579 SUMOylation controlled IA Gmax. The K579R mutation mimicked and occluded the SUMOylation-mediated decrease in IA Gmax, suggesting that SUMOylation at K579 blocked an intra- or inter-protein interaction involving K579. The K437R mutation did not obviously alter channel surface expression or biophysical properties, but it did block the SUMOylation-mediated increase in channel surface expression. Interestingly, enhancing K437 SUMOylation in the K579R mutant roughly doubled channel surface expression, but produced no change in IA Gmax, suggesting that the newly inserted channels were electrically silent. This is the first report that Kv4.2 channels are SUMOylated and that SUMOylation can independently regulate Kv4.2 surface expression and IA Gmax in opposing directions. The next step will be to determine if/how SUMOylation affects Kv4 interactions within the ternary complex.

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.

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

We have previously shown that androgens upregulate cardiac K(+) channels and shorten repolarization. However, the effects that estrogens (E2) and estrogen receptors (ER) might have on the various repolarizing K(+) currents and underlying ion channels remain incompletely understood. Accordingly, our objective was to verify whether and how E2 and its ERs subtypes influence these K(+) currents.

The rapidly activating and inactivating voltage-dependent outward K(+) (Kv) current, I(A), is widely expressed in central and peripheral neurons. I(A) has long been recognized to play important roles in determining neuronal firing properties and regulating neuronal excitability. Previous work demonstrated that Kv4.2 and Kv4.3 α-subunits are the primary determinants of I(A) in mouse cortical pyramidal neurons. Accumulating evidence indicates that native neuronal Kv4 channels function in macromolecular protein complexes that contain accessory subunits and other regulatory molecules. The K(+) channel interacting proteins (KChIPs) are among the identified Kv4 channel accessory subunits and are thought to be important for the formation and functioning of neuronal Kv4 channel complexes. Molecular genetic, biochemical, and electrophysiological approaches were exploited in the experiments described here to examine directly the roles of KChIPs in the generation of functional Kv4-encoded I(A) channels. These combined experiments revealed that KChIP2, KChIP3, and KChIP4 are robustly expressed in adult mouse posterior (visual) cortex and that all three proteins coimmunoprecipitate with Kv4.2. In addition, in cortical pyramidal neurons from mice lacking KChIP3 (KChIP3(-/-)), mean I(A) densities were reduced modestly, whereas in mean I(A) densities in KChIP2(-/-) and WT neurons were not significantly different. Interestingly, in both KChIP3(-/-) and KChIP2(-/-) cortices, the expression levels of the other KChIPs (KChIP2 and 4 or KChIP3 and 4, respectively) were increased. In neurons expressing constructs to mediate simultaneous RNA interference-induced reductions in the expression of KChIP2, 3, and 4, I(A) densities were markedly reduced and Kv current remodeling was evident.

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel-interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions.

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.

Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels.

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

BACKGROUND Transgenic mice (TG) with heart-directed overexpresion of the isoform of the transcription factor cyclic adenosine monophosphate response element modulator (CREM), CREM-IbΔC-X, display spontaneous atrial fibrillation (AF) and action potential prolongation. The remodeling of the underlying ionic currents remains unknown. Here, we investigated the regulatory role of CREM-IbΔC-X on the expression of K+ channel subunits and the corresponding K+ currents in relation to AF onset in TG atrial myocytes. METHODS AND RESULTS ECG recordings documented the absence or presence of AF in 6-week-old (before AF onset) and 12-week-old TG (after AF onset) and wild-type littermate mice before atria removal to perform patch clamp, contractility, and biochemical experiments. In TG atrial myocytes, we found reduced repolarization reserve K+ currents attributed to a decrease of transiently outward current and inward rectifier K+ current with phenotype progression, and of acetylcholine-activated K+ current, age independent. The molecular determinants of these changes were lower mRNA levels of Kcnd2/3, Kcnip2, Kcnj2/4, and Kcnj3/5 and decreased protein levels of K+ channel interacting protein 2 (KChIP2 ), Kir2.1/3, and Kir3.1/4, respectively. After AF onset, inward rectifier K+ current contributed less to action potential repolarization, in line with the absence of outward current component, whereas the acetylcholine-induced action potential shortening before AF onset (6-week-old TG mice) was smaller than in wild-type and 12-week-old TG mice. Atrial force of contraction measured under combined vagal-sympathetic stimulation revealed increased sensitivity to isoprenaline irrespective of AF onset in TG. Moreover, we identified Kcnd2, Kcnd3, Kcnj3, and Kcnh2 as novel CREM-target genes. CONCLUSIONS Our study links the activation of cyclic adenosine monophosphate response element-mediated transcription to the proarrhythmogenic electrical remodeling of atrial inward rectifier K+ currents with a role in action potential duration, resting membrane stability, and vagal control of the electrical activity.