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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON
1Department of Physiology and Biophysics, University at Buffalo-SUNY, Buffalo, New York; and 2Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China
Submitted 9 November 2005 ; accepted in final form 22 May 2006
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ABSTRACT |
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potassium channel inactivation; potassium channel ancillary subunits; closed-state inactivation; voltage-gated potassium channels
-subunits are integral membrane proteins that feature pore-forming and voltage-sensing domains (19). Additionally, it is likely that most, if not all, voltage-gated K+ channels exist in vivo as heteromeric protein complexes with one or more ancillary subunits (26, 35). Six major classes of voltage-gated K+ channel ancillary subunit have been described; these differ widely in their sequences, structures, effects on gating, tissue distribution, stoichiometry, and specificity of -subunit interaction (1, 16, 26, 27, 30, 52, 58). No sequence or structural motif identifies these proteins as K+ channel ancillary subunits, and many proteins homologous to K+ channel ancillary subunits perform other functions in the cell.
Two important classes of K+ channel ancillary subunit are the Kv channel interacting proteins (KChIPs) and dipeptidyl peptidases (DPPs), both of which are known for association with the Kv4 family. These channels produce transient K+ currents in response to membrane depolarization. They are responsible for important K+ currents in the heart, central nervous system, and smooth muscle (4). Ancillary subunits of Kv4 channels can be significant modulators of gating (4, 21, 35). The best characterized are the KChIPs (1). KChIPs are cytoplasmic proteins with four Ca2+-binding EF hands. In mammals, four KChIP genes express at least 21 mRNA splice variants, some of which may be species specific (8, 33, 36). When expressed with Kv4.2 or Kv4.3 in mammalian cell lines, KChIPs act as chaperones to increase cell surface expression through interaction with a conserved NH2-terminal domain of the Kv4 -subunit (47, 61). KChIPs can affect inactivation kinetics in a variety of ways but most accelerate recovery kinetics (21).
The DPP classes of K+ channel ancillary subunit, DPP6 and DPP101 , are related to the dipeptidyl aminopeptidase CD26/DPP4. DPP6 was established as a Kv4 ancillary subunit by copurification with Kv4.2 and KChIP1a from rat brain membranes (30). DPP10 was identified based on its sequence conservation with DPP6 (22). Like the KChIPs, DPP6 and DPP10 increase surface expression of Kv4.2 in heterologous expression systems through direct interaction with the -subunit. Both DPP6 and DPP10 accelerate inactivation and recovery from inactivation kinetics (20, 22, 30, 38, 42, 60). When expressed with the appropriate KChIP, Kv4.2 plus DPP6 or DPP10 closely mimics the properties of the native ISA in the central nervous system (CNS) (20, 30). Although it was originally reported to be localized primarily in the CNS (30, 55), DPP6 protein has been detected in human heart protein extracts, suggesting that it contributes to human cardiac transient outward current (Ito) (38) and perhaps to Kv4-based currents in other tissues.
Kv1.4 is another mammalian voltage-gated K+ channel with fast inactivation kinetics (40). It is widely distributed in the CNS where it is concentrated in axonal membranes within or near axon terminals (53). It is also present in cardiac ventricular endocardium, where it is responsible for a slowly recovering Ito (5). Kv1.4 is the mammalian channel whose mechanism of inactivation is most similar to the well-characterized Shaker K+ channel. Inactivation of Shaker and Kv1.4 is governed by N-type inactivation, where the NH2 terminus of the channel occludes the inner vestibule, and C-type inactivation, which involves external and internal pore closure (40). C-type inactivation is slower than N-type, is accelerated in the presence of N-type inactivation, and is the rate-limiting step in Kv1.4 recovery from inactivation (39). In Kv1.4 and other Kv1 channels, both types of inactivation can be enhanced by Kv
1 ancillary subunits (17, 29).
In light of this recent work, we wanted to determine whether, like other ancillary subunits, DPP10 could interact with -subunits outside the Kv4 family. DPP10 was tested with Kv1.4 because of the similarities noted between inactivation of Kv4 and Kv1.4 (12, 14, 23), which suggested the hypothesis that Kv1.4 gating could be modulated by DPP10. We found that expression of DPP10 and Kv1.4 in Xenopus laevis oocytes decreases time to peak current, causes negative shifts in the half-inactivation potential (V1/2) of steady-state activation and inactivation, and accelerates recovery from inactivation, all of which are similar to the consequences of coexpression of DPP10 and Kv4.3. The notable exception to this pattern was recovery from inactivation, which in Kv1.4 was slowed by coexpression with DPP10. We further explored DPP10 effects on inactivation by expression with KChIP2b and Kv4.3. Individually, each subunit caused a hyperpolarizing shift in steady-state inactivation, sped time to peak current, and increased the rate of recovery from inactivation. Expressed together, the effects of the two subunits were usually additive, except for recovery from inactivation, which was the same in the presence of either or both subunits. DPP10 and KChIP2b have very different effects on Kv4.3 closed-state inactivation. DPP10 enhances closed-state inactivation, whereas KChIP2b nearly abolishes it; expressed together, the DPP10 effect is clearly dominant. Deletion constructs of DPP10 expressed with Kv4.3 showed that the cytoplasmic NH2 terminus and transmembrane segment of DPP10 are sufficient for its modulation of Kv4.3 gating. These data suggest that DPP10 regulates Kv4.3 gating through a mechanism distinct from that of the KChIPs and that it might be a general regulator of voltage-gated K+ currents.
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MATERIALS AND METHODS |
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To construct mini-10, we utilized the plasmid pDPP10-N367, which encodes the first 367 amino acids of DPP10 and was a serendipitous artifact of the DPP10 cloning. Digestion of pDPP10-N367 with Acs I was followed by religation to form mini10, which contains only the coding sequence for the first 58 amino acids of DPP10, including the single transmembrane-spanning segment (see 
Fig. 6A).
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Electrophysiological techniques and data analyses.
Oocytes were clamped by a two-microelectrode bath clamp amplifier (OC-750A; Warner Instruments, Hamden, CT), as described previously (7, 51). Microelectrodes were fabricated from 1.5-mm-outer diameter borosilicate glass tubing with the use of a two-stage puller filled with 3 M KCl having resistances of 0.6–1.5 M
. During recording, oocytes were continuously perfused with control ND-96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, pH 7.4, adjusted with NaOH). Currents were recorded at room temperature (21–23°C) and filtered at 2.5 kHz.
Data were digitized and analyzed with pCLAMP 9 software (Molecular Devices, Sunnyvale, CA). Pulse protocols are described in the figure legends. Data are means ± SE. The time courses of both open- and closed-state inactivation were determined by fitting inactivation traces with the following function: f(t) = 
j=1NAjexp(–t/
j), where t is time, N = 1, 2, or 3, and Aj is the amplitude of jth component of inactivation (j = 1, 2, or 3). The kinetics of recovery from inactivation were determined by fitting recovery of the normalized peak current with the following function: frec(t) = 1 – j=1NBjexp(–t/recj), where N = 1 or 2 and Bj is the amplitude of jth component of recovery (rec) from inactivation (j = 1, 2). Steady-state inactivation was determined with the following fitting function: fi(V) = fi0/1 + exp[(V – V1/2)/k] + (1 – fi0), where fi0 is the constant and k is the slope. A fourth-power Boltzmann function, fa(V) = 1/{1 + exp[–(V – V1/2)/k]}4, was used for fitting steady-state activation.
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RESULTS |
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-subunits not considered to be their primary targets (4, 11, 21). Therefore, we tested DPP10 for its effects on another fast inactivating K+ channel, Kv1.4, and compared these results to those of DPP10 expressed with Kv4.3. Despite the resemblance in their inactivation time course, there are several important differences between Kv1.4 and Kv4.3. The amino acid sequence of Kv1.4 is only 22% conserved with Kv4.3, 39% in the core region inclusive of the membrane spanning regions. Kv1.4 recovery from inactivation is roughly 10 times slower than that of Kv4.3. Finally, although Kv1.4 displays "classic" N-type and C-type inactivation (7, 41), the inactivation in Kv4.3 is more complex and can proceed from both the open and closed states (23, 56). Despite these differences, similarities have been noted in Kv1.4 and Kv4.3 inactivation (12, 14, 23), suggesting the hypothesis that DPP10 might modulate Kv1.4 currents.
DPP10 and either Kv1.4 or Kv4.3 were coexpressed in Xenopus oocytes, and the resulting currents were measured by the two-electrode voltage-clamp technique. The results are summarized in Tables 1 and 2. Figure 1A shows superimposed raw current traces from clamp experiments of Kv1.4 and Kv1.4 + DPP10. Figure 1A, inset, shows that activation of Kv1.4 is more rapid in the presence of DPP10 than seen with the -subunit alone. Similar acceleration of activation is observed with Kv4.3 expressed with DPP10 (Fig. 1B) and is true for both channels at all voltages tested (Figs. 2A and 4A). Significant effects of DPP10 on Kv1.4 voltage dependence of steady-state inactivation and activation were also observed (Fig. 2, B and C; Table 1). DPP10 caused Kv1.4 steady-state inactivation to be shifted –8 mV, from a V1/2 of –52.3 ± 1.7 mV to –60.0 ± 0.6 mV (Fig. 2B), whereas the slope of steady-state inactivation voltage-dependence (k) decreased from 3.6 ± 0.1 mV to 3.0 ± 0.1 mV. Similar hyperpolarizing shifts were seen with steady-state activation (Fig. 2C). These shifts are similar to those caused by DPP10 when expressed with Kv4.3 (see Fig. 4, B and C).
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fast = 100 ± 9.7 ms, afast = 0.82 ± 0.03, and slow = 348.6 ± 35.8 ms (+50 mV). In the presence of DPP10, Kv1.4 inactivation is best described by three exponents: fast = 26.6 ± 1.8 ms, afast = 0.42 ± 0.06, intermediate = 108.1 ± 10.4 ms, aintermediate = 0.49 ± 0.05, and slow = 330.1 ± 31.7 ms (Fig. 3, A and B). The effects of DPP10 on Kv1.4 inactivation are reminiscent of interventions such as external pore mouth mutations and low external K+ concentration that accelerate C-type inactivation (40). Therefore, is it possible that DPP10 is also functioning through modulation of C-type inactivation.
One hallmark of the DPP10 effect on Kv4 channel gating is an acceleration of recovery from inactivation (20, 22, 42, 60). It is therefore somewhat surprising that DPP10 slows Kv1.4 recovery from inactivation. In the absence of DPP10, recovery of Kv1.4 is bi-exponential, with time constants of rec-fast = 930 ± 170 ms, arec-fast = 0.57 ± 0.04, and rec-slow = 4,430 ± 430 ms at –90 mV (Table 1). DPP10 increases the recovery time constants to rec-fast = 2,270 ± 230 ms, arec-fast = 0.30 ± 0.17, and rec-slow =10,980 ± 2,900 ms, resulting in a substantial slowing of recovery (Fig. 3, C and D). This slowing of recovery from inactivation is a major difference between the effects of DPP10 on Kv1.4 and Kv4.3 (compare to Fig. 5D).
To ensure that the effect of DPP10 on Kv1.4 was specific for some of the K+ channels and not a general effect of DPP10 on K+ channels, we expressed DPP10 with a closely related voltage-gated K+ channel, Kv1.1. Normalized raw current traces, the current-voltage relationship (Fig. 1, C and D), steady-state activation, and time to peak current (not shown) show that DPP10 has no detectable effect on Kv1.1 gating and kinetics. Together, these data show that DPP10 is capable of specific modulation of Kv1.4 currents expressed in Xenopus oocytes.
DPP10 expression with Kv4.3 and KChIP2b. To understand further the physiological consequences of DPP10 expression, we expressed it in the presence of Kv4.3 or Kv4.3 and KChIP2b. We chose to express these proteins in Xenopus oocytes to allow us to assess the effects of KChIP2b and DPP10 expression in the absence of potentially confounding K+ channel subunits (33).
Figure 4D shows superimposed raw traces from two-electrode voltage-clamp experiments of Kv4.3 + KChIP2b and Kv4.3 + DPP10 + KChIP2b at +50 mV; controls without KChIP2b are shown in Fig. 1B. The effects of KChIP2b on Kv4.3 activation are negligible at +50 mV and become manifest at voltages less than –10 mV. The effects of DPP10 are more striking, as DPP10 accelerates Kv4.3 activation in both the absence and presence of KChIP2b. This is true at all voltages tested (Fig. 4A; Table 2).
In contrast to DPP10, KChIP2b does not change the V1/2 or k of the Kv4.3 current steady-state activation significantly (Fig. 4C; Table 2). Coexpression of KChIP2b and Kv4.3 with DPP10 gave a V1/2 nearly identical to Kv4.3 expressed alone, a value between that caused by either KChIP2b or DPP10 when expressed with Kv4.3. We also examined the effect of DPP10 on steady-state inactivation of Kv4.3 or Kv4.3 + KChIP2b (Fig. 4B). Although DPP10 alone produces a large hyperpolarizing shift of V1/2 and decrease in k of Kv4.3 steady-state inactivation, DPP10 with KChIP2b caused a smaller hyperpolarizing shift in V1/2 and a negligible change in k (Table 2, Fig. 4B).
DPP10 and KChIP2b have complex effects on Kv4.3 inactivation kinetics. The major distinguishing feature of Kv4 inactivation is that a substantial portion can occur from the closed state (2, 3, 23, 56), unlike Shaker and Kv1.4 where inactivation proceeds primarily from the open state (40). Our group (56) recently showed that inactivation in Kv4.3 occurred both from closed and open states. Our model of Kv4.3 gating incorporated inactivation from both closed and open states and allowed direct communication between the open-inactivated and closed-inactivated states (56).
Figure 5, A and B, shows that Kv4.3 open-state inactivation is a tri-exponential process, dominated by the fastest time constant at positive voltages (56). KChIP2b increases the dominance of the fastest time constant such that inactivation kinetics are best described by a mono-exponential function with a time constant of 89.1 ± 3.9 ms at +50 mV, roughly intermediate between fast and intermediate of Kv4.3 alone. In contrast, DPP10 expressed with Kv4.3 decreased fast and intermediate to 17.7 ± 2.0 and 88.3 ± 13.1 ms, respectively, in both cases roughly twice as fast as the fast and intermediate time constants of Kv4.3 expressed alone. When DPP10 was coexpressed with KChIP2b and Kv4.3, the inactivation time course became tri-exponential. However, inactivation was different from that of Kv4.3 and Kv4.3 + DPP10 in that slow was faster and had a much larger amplitude at the most positive potentials: a = 0.46 ± 0.02 vs. 0.11 ± 0.01 with Kv4.3 + DPP10 and 0.16 ± 0.01 with Kv4.3 (at +50 mV).
DPP10 has a dramatic effect on closed-state inactivation (Fig. 5C; Table 2), showing maximal inactivation within 4 s at –60 mV. This represents a substantial hyperpolarizing shift in voltage dependence; the time course is typical of Kv4.3 closed-state inactivation at –40 mV (56). Interestingly, closed-state inactivation in the presence of KChIP2b shows little development at –60 mV. Although the time constant of closed-state inactivation of Kv4.3 + KChIP2b is faster than Kv4.3, only 13% of Kv4.3 + KChIP2b channels have inactivated through this pathway by 10 s. This correlates with the higher voltage sensitivity of Kv4.3 current inactivation in the presence of KChIP2b (Fig. 4B; note the small amount of inactivation at –60 mV). In contrast, the steady-state inactivation V1/2 of Kv4.3 + DPP10, –59.7 ± 1.3 mV, is nearly equal to the V1/2 that we determined for Kv4.3 closed-state inactivation (56), suggesting that DPP10 causes a significant modulation of Kv4.3 inactivation through enhancement of closed-state inactivation. Coexpression of DPP10, KChIP2b, and Kv4.3 results in closed-state inactivation kinetics that fall between the rate of Kv4.3 expressed with each of the two subunits alone, although the extent of closed-state inactivation is clearly much closer to that seen with Kv4.3 + DPP10, suggesting the DPP10 effect dominates closed-state inactivation.
A common hallmark of Kv4-dependent fast-inactivating K+ currents in vivo is recovery from inactivation faster than that observed with Kv4.3 in the absence of ancillary subunits. As previously shown, both DPP10 and KChIP2b accelerate recovery from inactivation (Fig. 5D), although with slightly different kinetics. Together, KChIP2b and DPP10 have recovery kinetics that do not differ significantly from those with KChIP2b alone.
The extracellular portion of DPP10 is dispensable. The DPP10 paralogs, DPP6 and DPP4, form dimers (13, 50). The similarity between these proteins and DPP10 suggests that these also form dimers. The observation that the bulk of the protein (730 of 796 amino acids) is extracellular and its functionality suggest an important role for the external domain. However, most K+ channel kinetic modulation factors function from the intracellular side of the channel. To test the portions of DPP10 responsible for modulation of Kv4.3 activity, we constructed a series of deletions from the COOH terminus of DPP10 and expressed them with Kv4.3. The largest deletion was "mini-10," a construct containing the 34 amino acid cytoplasmic NH2 terminus, the 22 amino acid membrane-spanning segment, and 2 amino acids predicted to be extracellular (Fig. 6A).
Experiments to test the functionality of the construct were performed as in Figs. 4 and 5 by coexpression of deletion constructs, including mini-10, and Kv4.3 in Xenopus oocytes. The results of the deletions were identical (data not shown). The results of coexpression of mini-10 and Kv4.3 are presented in Fig. 6. The current-voltage relationship (Fig. 6B) and time to peak current (Fig. 6C) and influence on steady-state inactivation (Fig. 6E) are nearly identical to full-length DPP10 + Kv4.3, whereas the steady-state activation shift is slightly larger in the presence of mini-10 (Fig. 6D). Finally, deletion of the DPP10 extracellular domain speeds recovery from inactivation (Fig. 6F), although to a lesser degree than full-length DPP10. Together, it is clear that all the functionality of DPP10 is located within the cytoplasmic and transmembrane segments; therefore, dimerization is not required for modulation of Kv4.3 kinetics.
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DISCUSSION |
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Our most surprising finding was that DPP10 had profound influence on Kv1.4 gating properties. DPP10 hyperpolarized the V1/2 and increased voltage sensitivities (decreased k) of both steady-state activation and steady-state inactivation. It also sped inactivation kinetics. In these respects, DPP10 modulation of Kv1.4 is similar to that of Kv4.3. However, DPP10 slowed Kv1.4 recovery from inactivation. These observations are consistent with previous views of Kv1.4 inactivation, which occur through the classic N- and C-type inactivation mechanisms first described for Shaker (40). Although in many ways Kv4 inactivation is different from that of Kv1.4, similarities have been reported. For example, some components of Kv4.2 inactivation bear important hallmarks of N-type inactivation (14). It has also been suggested that Kv4.3 may undergo C-type inactivation (12). However, Kv4.3 differs from Kv1.4 in that there are large differences in the voltage dependence of inactivation, a reflection of different mechanisms of coupling inactivation to activation (56). In contrast to Kv4.3 inactivation mechanisms, Kv1.4 N- and C-type inactivation are coupled to each other and have the same coupling to activation (40).
The striking slowing of Kv1.4 recovery from inactivation and acceleration of inactivation kinetics caused by DPP10 are reminiscent of the effects seen with Kv1.2 (6, 28, 29), a cytoplasmic ancillary subunit of Kv1 channels. Kv1.2 increases the rate of Kv1.4 inactivation through an increase in the rate of C-type inactivation. However, unlike the intrinsic C-type inactivation well characterized in Kv1.4 and other channels, where many critical amino acid determinants are in the external pore (40), this was due to interaction with the unique cytoplasmic NH2 terminus of Kv1.2. It was proposed that, like the NH2 terminus of Kv1.1, which causes N-type inactivation through its NH2-terminal "ball" domain (43), the Kv1.2 NH2 terminus could interact with the inner vestibule of the pore. This interaction has low affinity and rapid kinetics such that it cannot cause appreciable N-type inactivation like Kv1.1, but it enhanced C-type inactivation through coupling with N-type inactivation (29). Because C-type inactivation is the rate-limiting step in Kv1.4 recovery (39), enhancement of C-type inactivation slows overall recovery from inactivation, as seen with DPP10. This finding is of broad significance toward understanding K+ channel inactivation, as modulation of both channel types by DPP10 suggests that there are common motifs within Kv4.3 and Kv1.4 that are responsible for DPP10 activity.
Our Kv1.4 data are in apparent conflict with Ren et al. (42), who were able to coimmunoprecipitate tagged Kv4.3 and DPP10 heterologously expressed in HEK-293 cells but not tagged Kv4.3 and Kv1.4. There are several potential explanations for this discrepancy. Difficulties in immunoprecipitation of Kv4/DPP complexes have been noted (20, 60), suggesting that Kv1.4-DPP10 interactions may be difficult to detect. Ren et al. performed their experiments in HEK-293 cells, whereas ours were performed in Xenopus oocytes. Important differences in K+ channel expression have been noted between oocytes and mammalian cell lines (20). It is possible that the HEK-293 cells express a protein that interferes with Kv1.4-DPP10 interaction. Conversely, it is also possible that a factor present in oocytes and not HEK-293 cells is required for interaction. Finally, our data do not show direct interaction between the proteins. DPP10 could affect a signaling pathway in oocytes that modifies Kv1.4 gating through a pathway entirely different from that of Kv4.3.
A second major finding of this study was the effect of KChIP2b and DPP10 on closed-state inactivation of Kv4.3, showing that it was stimulated by DPP10 and inhibited by KChIP2b. These effects can be understood by looking at the shifts in steady-state activation and inactivation (Fig. 4). DPP10 causes a –10-mV shift in steady-state activation, suggesting that it reduces the energy barrier for channel opening. However, the shift moves V1/2 closer to the range where closed-state inactivation predominates (56), thus favoring closed-state inactivation. Similar effects have been seen on coexpression of Kv4.2 and DPP10 (22).
The disparate effects of DPP10 and KChIP2b on Kv4.3 can be understood in terms of their effect on gating transitions. DPP10 accelerated the forward processes of activation and inactivation, as well as a reverse process, recovery from inactivation. This suggests that DPP10 affects gating transitions by lowering the energy barrier in both forward and reverse transitions. KChIP2b increases the energy barrier for some forward transitions, resulting in, for example, the slowing of open-state inactivation and reduction in the development of closed-state inactivation. However, like DPP10, KChIP2b also reduces the barrier for the backward transition of recovery from inactivation, as has been suggested (34). The DPP10-KChIP2b coexpression experiments support this view. When expressed together, forward processes tend to give an intermediate effect, whereas recovery from inactivation retains the faster rate seen in the presence of KChIP2b alone. Therefore, the role of DPP10 and KChIP2b could be to fine tune inactivation kinetics and determine the Kv4.3 inactivation pathway, while preserving the fast recovery from inactivation normally seen in Kv4-based currents in vivo, an effect that could have profound physiological implications (56).
Recently, the kinetic effects of coexpression of DPP10 with Kv4.2 and KChIP3 were examined (20). Comparison of our results with this study are difficult, not only because of differences in Kv4.2 + KChIP3 and Kv4.3 + KChIP2b kinetics (20, 34, 57) but also because of the differences in gating of the -subunits. However, comparisons of our results with those of Jerng et al. (20) are instructive. DPP10 dominated the inactivation time course of the Kv4.2 + KChIP3 complex, whereas its effects on Kv4.3 + KChIP2b inactivation kinetics were subtle, with neither ancillary subunit clearly dominant (Fig. 5). Recovery from inactivation was faster in the Kv4.2-DPP10-KChIP3 complex than it was with either ancillary subunit alone. We found that this was not the case with Kv4.3, as recovery in the presence of Kv4.3 plus DPP10 and KChIP2b was roughly the same as with KChIP2b alone. The shifts in steady-state inactivation and in time to peak current were similar in both cases (Fig. 4). Examination of the effect of KChIP3 on Kv4.2 shows that this subunit does not increase the rate of recovery to the same extent in Kv4.2 as KChIP2b does in Kv4.3. Additionally, KChIP3 has a dramatic influence on Kv4.2 inactivation kinetics, slowing them much more than is seen with KChIP2b associated with Kv4.3. Although differences in Kv4.3 and Kv4.2 gating cannot be definitively ruled out, the most likely explanation for the differences between Jerng et al. (20) and this work are primarily because of the differing effects of the KChIPs.
Considerable structural information is available for DPP6 and DPP10, both from the direct structural determination of the DPP6 extracellular domain (50) and because of their relationship to the intensively studied DPP4 protease (18), which has no activity toward Kv4 channels (22, 42, 60). DPP10 and DPP6 are type II membrane-spanning proteins with roughly 740 of their 800 amino acids (796 and 804 amino acids for DPP10 and DPP6, respectively) extracellular. The external domain of DPP6 is critical for dimerization (50). DPP10 and DPP6 lack the activity found in DPP4 (13, 37, 50). There are two forms of DPP6 that differ in their NH2-terminal cytoplasmic region; only the short form (802 amino acids) modulates Kv4 (22). The cytoplasmic NH2 termini and single transmembrane segment of the short DPP6 and DPP10 have 58 nonidentical amino acids. Several lines of evidence suggest that the short cytoplasmic NH2 termini are responsible for modulation of Kv4 gating (22, 42, 42, 60); however, it is not clear whether this region could replicate all the functions of the full-length proteins; in addition, the role of DPP dimerization was not addressed.
Our data show that only 58 amino acids of DPP10 are responsible for the modulation of Kv4.3 gating. Dimerization is clearly not required for DPP10 function, as we have found that the NH2-terminal 34 amino acids and transmembrane segment are capable of replicating the gating effects of the full-length DPP10 on Kv4.3 and Kv4.3 + KChIP2b. In this regard, our data are similar to data previously described that suggested that these regions of DPP10 and DPP6 could interact directly with Kv4.2 (22, 60) or Kv4.3 (42). Our data differ in that we do not use chimeric DPPs (22, 42) and we use a more extensive set of gating analysis, arguing strongly that all the amino acids required for DPP10-mediated modification of Kv4.3 gating are present in the transmembrane-spanning region and NH2 terminus.
This is the first study of DPP10 that focuses on the effects on Kv4.3 expressed with KChIP2b. It is presently unknown whether this exact combination of three proteins exists in vivo. However, the association between DPP10 and Kv4.3 has been clearly established in native tissue by reciprocal coimmunoprecipitation of the proteins from rat brain extracts (30). KChIP2b is more complex because it is one of at least 10 splice variants of KChIP2 (8, 33, 36). In general, localization studies have used antibodies that would cross-react with many of the KChIP2 splice forms; however, at least one KChIP2 splice form colocalizes with Kv4.3 and DPP10 in neocortical layers II-VI and the dentate gyrus (1, 20, 44, 48, 59). Although KChIP2b seems to be typical of KChIP2 activity toward Kv4.3 (9–11, 15, 34, 57), some splice variants have much different activity (8). The results presented here should be interpreted with these observations in mind.
DPP10-based modulation of Kv1.4 is a potentially important mechanism for regulation of K+ currents. Kv1.4 channels are widely distributed in the CNS and peripheral nervous system, where they are concentrated along axons and axonal membranes at axon termini, suggesting that they play a role in neurotransmitter release though regulation of nerve terminal membrane potential (53). In the CNS, they have been shown to colocalize in several regions (45). Kv1.4 is also present in mammalian hearts, where it is responsible for a slowly recovering Ito found in the left ventricular endocardium (5, 32, 49). DPP10 modulation of Kv1 might not be restricted to Kv1.4, as other members of this family undergo similar inactivation (40), suggesting a broad range of potential roles for DPP10.
Association of DPP10 with transiently inactivating K+ currents could have profound physiological implications. For example, cardiac myocytes have resting potentials of –80 to –90 mV at normal extracellular K+ concentrations. Under these conditions, a transient depolarization (<100 ms, similar to the duration of a human atrial action potential) to +30 mV will result in open-state inactivation in the absence of ancillary subunits. Closed-state inactivation would be more of a factor if DPP10 were present. However, during acute myocardial ischemia, extracellular K+ can rise to 12 mM, increasing resting potential closer to –65 mV (31). These conditions would favor closed-state inactivation, rendering Kv4.3 unavailable (56), a situation that would be exacerbated by DPP10. In contrast, neurons have more positive resting potentials than cardiac myocytes (24). This would lead to a large degree of closed-state inactivation of Kv4.3-based currents in the presence of DPP10, thus limiting the availability of Kv4 channels to contribute to regulation of neuronal spiking.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 DPP6 is also known as DPPX and DPP10 as DPPY (55). A recently proposed nomenclature (20) suggests the names DPL6 and DPL10, respectively, for dipeptidyl peptidase-like proteins, reflecting their lack of protease activity. 
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) and Kv1.1 expressed with DPP10 (
). Pulse protocol: from a holding potential of –90 mV, 15-s initial pulses were applied to +50 mV in 10-mV steps (n = 5).
, Kv4.3 + KChIP2b; 
, Kv4.3 + KChIP2b + DPP10. A: time to peak current as a function of voltage decreases in the presence of DPP10. B: DPP10 causes a hyperpolarizing shift in steady-state inactivation. Protocols for A and B were described in 
, Kv4.3 + mini-10. B-F: peak current-voltage relationships, time to peak currents, steady-state activation, steady-state inactivation, and recovery from inactivation were all performed with corresponding voltage-clamp protocols as described in