DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4

Voltage-gated K⁺ channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K⁺ currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At –60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K⁺ channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K⁺ channels. potassium channel inactivation; potassium channel ancillary subunits; closed-state inactivation; voltage-gated potassium channels     

Modulation of Kv4.2 channel expression and gating by dipeptidyl peptidase 10 (DPP10)

The dipeptidyl aminopeptidase-like protein DPPX (DPP6) associates with Kv4 potassium channels, increasing surface trafficking and reconstituting native neuronal ISA-like properties. Dipeptidyl peptidase 10 (DPP10) shares with DPP6 a high amino acid identity, lack of enzymatic activity, and expression predominantly in the brain. We used a two-electrode voltage-clamp and oocyte expression system to determine if DPP10 also interacts with Kv4 channels and modulates their expression and function. Kv4.2 coimmunoprecipitated with HA/DPP10 from extracts of oocytes heterologously expressing both proteins. Coexpression with DPP10 and HA/DPP10 enhanced Kv4.2 current by approximately fivefold without increasing protein level. DPP10 also remodeled Kv4.2 kinetic and steady-state properties by accelerating time courses of inactivation and recovery (taurec: WT = 200 ms, +DPP10 = 78 ms). Furthermore, DPP10 introduced hyperpolarizing shifts in the conductance-voltage relationship (approximately 19 mV) as well as steady-state inactivation (approximately 7 mV). The effects of DPP10 on Kv4.1 were similar to Kv4.2; however, distinct biophysical differences were observed. Additional experiments suggested that the cytoplasmic N-terminal domain of DPP10 determines the acceleration of inactivation. In summary, DPP10 is a potent modulator of Kv4 expression and biophysical properties and may be a critical component of somatodendritic ISA channels in the brain.     

Augmentation of Kv4.2-encoded currents by accessory dipeptidyl peptidase 6 and 10 subunits reflects selective cell surface Kv4.2 protein stabilization

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.     

I SA channel complexes include four subunits each of DPP6 and Kv4.2

Kv4 potassium channels produce rapidly inactivating currents that regulate excitability of muscles and nerves. To reconstitute the neuronal A-type current I(SA), Kv4 subunits assemble with DPP6, a single transmembrane domain accessory subunit. DPP6 alters function-accelerating activation, inactivation, and recovery from inactivation-and increases surface expression. We sought here to determine the stoichiometry of Kv4 and DPP6 in complexes using functional and biochemical methods. First, wild type channels formed from subunit monomers were compared with channels carrying subunits linked in tandem to enforce 4:4 and 4:2 assemblies (Kv4.2-DPP6 and Kv4.2-Kv4.2-DPP6). Next, channels were overexpressed and purified so that the molar ratio of subunits in complexes could be assessed by direct amino acid analysis. Both biophysical and biochemical methods indicate that I(SA) channels carry four subunits each of Kv4.2 and DPP6.     

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells

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.     

A New TASK for Dipeptidyl Peptidase-like Protein 6

Dipeptidyl Peptidase-like Protein 6 (DPP6) is widely expressed in the brain where it co-assembles with Kv4 channels and KChIP auxiliary subunits to regulate the amplitude and functional properties of the somatodendritic A-current, I_SA. Here we show that in cerebellar granule (CG) cells DPP6 also regulates resting membrane potential and input resistance by increasing the amplitude of the I_K(SO) resting membrane current. Pharmacological analysis shows that DPP6 acts through the control of a channel with properties matching the K_2P channel TASK-3. Heterologous expression and co-immunoprecipitation shows that DPP6 co-expression with TASK-3 results in the formation of a protein complex that enhances resting membrane potassium conductance. The co-regulation of resting and voltage-gated channels by DPP6 produces coordinate shifts in resting membrane potential and A-current gating that optimize the sensitivity of I_SA inactivation gating to subthreshold fluctuations in resting membrane potential.     

Alternative splicing of the rat Ca(v)3.3 T-type calcium channel gene produces variants with distinct functional properties(1)

Molecular diversity in T-type Ca(2+) channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Ca(v)3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Ca(v)3.3 gene produces channels with distinct properties.     

DPP10 modulates Kv4-mediated A-type potassium channels

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase (DPP) IV known as DPP10 was recently identified and linked to asthma susceptibility; however, the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels (Nadal, M. S., Ozaita, A., Amarillo, Y., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) Neuron. 37, 449-461). We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to DPPX activity and significantly different from DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude, and modified the voltage dependence and kinetic properties of the current such that they resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization, we found DPP10 to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming the association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10, and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity.     

DPP6 Domains Responsible for Its Localization and Function

Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K⁺ channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.     

N-glycosylation of the mammalian dipeptidyl aminopeptidase-like protein 10 (DPP10) regulates trafficking and interaction with Kv4 channels

The dipeptidyl aminopeptidase-like protein 10 (DPP10) is a type II transmembrane protein homologue to the serine protease DPPIV/CD26 but enzymatically inactive. In the mammalian brain, DPP10 forms a complex with voltage-gated potassium channels of the Kv4 family, regulating their cell surface expression and biophysical properties. DPP10 is a glycoprotein containing eight predicted N-glycosylation sites in the extracellular domain. In this study we investigated the role of N-glycosylation on DPP10 trafficking and functional activity. Using site-directed mutagenesis (N to Q) we showed that N-glycosylation occured at six positions. Glycosylation at these specific residues was necessary for DPP10 trafficking to the plasma membrane as observed by flow cytometry. The surface expression levels of the substitutions N90Q, N119Q, N257Q and N342Q were reduced by more than 60%. Hence the interaction with the Kv4.3/KChIP2a channel complex was disrupted preventing the hastening effect of wild type DPP10 on current kinetics. Interestingly, N257 was crucial for this function and its substitution to glutamine completely blocked DPP10 sorting to the cell surface and prevented DPP10 dimerization. In summary, we demonstrated that glycosylation was necessary for both DPP10 trafficking to the cell surface and functional interaction with Kv4 channels.     

KCNE1 constrains the voltage sensor of Kv7.1 K+ channels

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K(+) channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K(+) channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions.     

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.     

Kv4.2 and Accessory Dipeptidyl Peptidase-like Protein 10 (DPP10) Subunit Preferentially Form a 4:2 (Kv4.2:DPP10) Channel Complex

Kv4 is a member of the voltage-gated K⁺ channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.     

Rate-limiting Reactions Determining Different Activation Kinetics of Kv1.2and Kv2.1 Channels

To identify the mechanisms underlying the faster activation kinetics in Kv1.2 channels compared to Kv2.1 channels, ionic and gating currents were studied in rat Kv1.2 and human Kv2.1 channels heterologously expressed in mammalian cells. At all voltages the time course of the ionic currents could be described by an initial sigmoidal and a subsequent exponential component and both components were faster in Kv1.2 than in Kv2.1 channels. In Kv1.2 channels, the activation time course was more sigmoid at more depolarized potentials, whereas in Kv2.1 channels it was somewhat less sigmoid at more depolarized potentials. In contrast to the ionic currents, the ON gating currents were similarly fast for both channels. The main portion of the measured ON gating charge moved before the ionic currents were activated. The equivalent gating charge of Kv1.2 ionic currents was twice that of Kv2.1 ionic currents, whereas that of Kv1.2 ON gating currents was smaller than that of Kv2.1 ON gating currents. In conclusion, the different activation kinetics of Kv1.2 and Kv2.1 channels are caused by rate-limiting reactions that follow the charge movement recorded from the gating currents. In Kv1.2 channels, the reaction coupling the voltage-sensor movement to the pore opening contributes to rate limitation in a voltage-dependent fashion, whereas in Kv2.1 channels, activation is additionally rate-limited by a slow reaction in the subunit gating.     

Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C termini

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K(+) channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta2 subunits in the brain. Expression of Kvbeta1 or Kvbeta2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta-associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K⁺ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.