Modulation of Kv4-encoded K(+) currents in the mammalian myocardium by neuronal calcium sensor-1.

Voltage-gated K(+) channels are multimeric proteins, consisting of four pore-forming alpha-subunits alone or in association with accessory subunits. Recently, for example, it was shown that the accessory Kv channel interacting proteins form complexes with Kv4 alpha-subunits and modulate Kv4 channel activity. The experiments reported here demonstrate that the neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily, is expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from (adult mouse) ventricular extracts. In addition, co-expression studies in HEK-293 cells reveal that NCS-1 increases membrane expression of Kv4 alpha-subunits and functional Kv4-encoded K(+) current densities. Co-expression of NCS-1 also decreases the rate of inactivation of Kv4 alpha-subunit-encoded K(+) currents. In contrast to the pronounced effects of Kv channel interacting proteins on Kv4 channel gating, however, NCS-1 co-expression does not measurably affect the voltage dependence of steady-state inactivation or the rate of recovery from inactivation of Kv4-encoded K(+) currents. Taken together, these results suggest that NCS-1 is an accessory subunit of Kv4-encoded I(to,f) channels that functions to regulate I(to,f) density in the mammalian myocardium.

The rapidly activating and inactivating depolarization-activated fast transient outward K ϩ current, I to,f , underlies the rapid initial phase of action potential repolarization in mammalian cardiac cells, and heterogeneities in I to,f expression contribute to observed variations in action potentials waveforms in different regions of the heart (1,2). In addition, there are changes in the expression and the properties of I to,f during normal cardiac development (3) and in a variety of myocardial disease states (2,4,5). Changes in I to,f density can result in marked alterations in action potential waveforms, influence the normal propagation of activity in the myocardium, and increase the susceptibility to lethal cardiac arrhythmias (2,4,5). Thus, there is considerable interest in defining the molecular correlates of functional I to,f channels and in delineating the molecular mechanisms controlling I to,f expression in the mammalian myocardium. Importantly, considerable evidence has accumulated demonstrating that pore-forming ␣-subunits of the Kv4 subfamily encode cardiac I to,f channels (6 -10). In contrast, the role of accessory subunits in the generation of functional I to,f channels in the mammalian heart is poorly understood (1).
The experiments reported here were focused on examining the expression and the functioning of neuronal calcium sensor protein-1 (NCS-1), another member of the recoverin-neuronal calcium sensor superfamily (21), in the mammalian myocardium. Biochemical data are presented demonstrating that NCS-1 is highly and uniformly expressed in adult mouse ventricles and that NCS-1 co-immunoprecipitates with Kv4.3 from mouse ventricular extracts. In addition, co-expression studies revealed that NCS-1 increases functional Kv4-encoded K ϩ cur-rent densities and decreases the rate of current inactivation. Taken together, these observations suggest that NCS-1 is an integral component of cardiac I to,f channels. A preliminary account of some of the findings presented here has appeared previously in abstract form (22).

EXPERIMENTAL PROCEDURES
Western Blots-Lysates and membrane preparations were prepared from adult (6 -8 week) C57BL6 mouse brains, ventricles, and isolated ventricular myocytes as well as from Kv4.3-expressing HEK-293 cells, using methods described previously (23,24). The protein content of each of the solubilized samples was determined by using a Bio-Rad protein assay kit with bovine serum albumin as a standard. Proteins were fractionated on 8 -15% SDS-PAGE gels and transferred to PVDF membranes. The PVDF membrane strips were then incubated in 0.2% I-Block (Tropix) in PBS containing 0.1% Tween 20 (blocking buffer) for 1 h at room temperature, followed by overnight incubations at 4°C with a specific polyclonal anti-Kv4.3 antibody, anti-Kv4.3a (20), or a polyclonal anti-NCS-1 antibody (25) at 5 g/ml. After washing, the membrane strips were incubated for 2 h at room temperature with alkaline phosphatase-conjugated goat anti-rabbit IgG (Tropix) diluted 1:5000 in the blocking buffer, and bound antibodies were detected using the CPSD chemiluminescent alkaline phosphate substrate (Tropix).
Immunoprecipitations-Immunoprecipitations were performed with a polyclonal anti-Kv4.3 antibody (Alomone), anti-Kv4.3b, as described previously (20). The anti-Kv4.3b antibody (0.5 g) was incubated with equilibrated protein A-Sepharose beads (50 l) (Sigma) prior to the addition of aliquots (100 l) of mouse ventricular lysates. After mixing (by inversion) overnight at 4°C, precipitating material was collected by centrifugation and extracted by heating in PBS containing 1% SDS. The resulting protein samples were fractionated on SDS-PAGE gels and subjected to immunoblot analysis with either the polyclonal anti-Kv4.3a (20) or the anti-NCS-1 (25) antibody. Following exposure to secondary antibody, protein bands were visualized by enhanced chemiluminescence as described above.
Construction of Adenoviral Vectors-The recombinant adenoviral vectors, AdEGI-Kv4.2 and AdEGI-Kv4.3, were generated by Cre-lox recombination, as reported previously (26,27). The coding sequence for mKv4.2 or mKv4.3 was cloned into the adenovirus shuttle vector pAdEGI (26,27). Because polycistronic vectors result in an Ϸ3:1 ratio of enhanced green fluorescent protein (EGFP) to ion channel protein (28), infected cells can be readily identified under epifluorescence illumination (20). The adenoviral vector AdVgRXR, encoding the ecdysone receptor, was generated by subcloning the expression cassette from pVgRXR (Invitrogen) into pAdLox. The replication-defective recombinant NCS-1 (and KChIP2) adenoviruses were constructed using methods described previously (29). Briefly, the cDNA encoding wild-type (rat) NCS-1 (or KChIP2) was subcloned into a modified version of the pXCRSV bicistronic shuttle vector that also expresses EGFP. The resulting pXCRSV-NCS-1 (or pXCRSV-KChIP2) was then co-transfected with pBHG11ts into HEK-293 cells. Successful recombinants were identified by the cytopathic effect on HEK-293 cells and EGFP fluorescence. Transgene expression was also confirmed by Western blot analysis. Following PCR screening, recombinant viruses were plaque-purified and examined for replication-competent adenoviruses by PCR (30). Plaque-purified viruses were then amplified and further purified by double cesium chloride (CsCl) gradient ultracentrifugation. The number of virus particles was determined spectrophotometrically, and the number of infectious particles was estimated by crystal violet staining using the agarose overlay method; virus concentrations were 10 9 -10 10 plaque-forming units (PFUs) per ml.
Cell Lines and Adenoviral Infections-HEK-293 cells, obtained from the Washington University Medical School Tissue Culture Support Center, were maintained in Opti-MEM (Invitrogen), supplemented with 5% horse serum, 5% heat-inactivated fetal calf serum, and 1 unit/ml penicillin/streptomycin. Cells were passaged at confluence (every 3-4 days) by brief trypsinization. In some experiments, HEK-293 cells were transiently transfected with plasmids encoding Kv4.2, Kv4.3, and Kv2.1 alone or in combination with NCS-1 or KChIP2 (and EGFP) by using the calcium-phosphate precipitation method. Approximately 16 h after the transfections, the cells were washed and allowed to recover for 20 -24 h prior to electrophysiological recordings.
For adenoviral infections, HEK-293 cells were transferred (ϳ48 h after passaging) into fresh Opti-MEM containing either the AdEGI-Kv4.2 or the AdEGI-Kv4.3 adenovirus (at 500 PFU/cell), the ecdysone receptor virus (at 50 PFU/cell), and 2% fetal calf serum. After 2 h, the infection medium was replaced with growth medium. Approximately 24 h later, cells were secondarily infected with either the NCS-1 virus (or the KChIP2 virus) at 500 PFU/cell. Kv4.x expression was then induced by addition of 5 M muristerone A, and electrophysiological recordings were obtained ϳ20 -24 h later.
Antisense Experiments-For antisense experiments, HEK-293 cells stably expressing Kv4.3 2 were maintained in Opti-MEM selection medium, supplemented with 5% horse serum, 5% fetal calf serum, 1 unit/ml penicillin/streptomycin, and 300 g/ml geneticin (Sigma). Antisense oligodeoxynucleotides (AsODNs) targeted against the translation start sites (nucleotides 4 -23) of human NCS-1 (AsODN-NCS-1, 5Ј-AAC TTG CTG TTG GAT TTC CC-3Ј) and a randomized control AsODN (5Ј-CAT CTT TAG AGC TGG TCC TT-3Ј) were obtained from Integrated DNA Technologies, Inc. These AsODNs were synthesized with a phosphorothioate backbone and tagged at the 5Ј end with Cy3 to allow visualization of uptake. For experiments, AsODNs (2 M per 35-mm dish) were mixed with LipofectAMINE (2 g/ml; Invitrogen) and incubated at room temperature for 15 min prior to addition of the LipofectAMINE/AsODN mixture to the cells. After 12 h, the AsODNcontaining medium was removed and replaced with selection medium, and the cells were harvested 24 -36 h later.
To determine the rates of recovery from steady-state inactivation, cells were first depolarized to ϩ50 mV for 400 ms to inactivate the currents (conditioning pulses), subsequently hyperpolarized to Ϫ70 or Ϫ90 mV for varying times ranging from 5 ms to 2 s, and finally stepped to ϩ50 mV for 400 ms (test pulses) to activate the currents and assess the extent of recovery. The voltage dependence of steady-state inactivation were also examined using a two-pulse protocol; 1-s prepulses to potentials between Ϫ110 and ϩ30 mV followed by a 400-ms test pulse to ϩ50 mV were used. Experimental data were acquired at variable sampling frequencies, and current signals were filtered on-line at 5 kHz prior to digitization and storage.
Data Analysis-Analysis of electrophysiological data was completed using Clampfit 6.0.5 (Axon). The decay phases of the voltage-activated, Kv4.x-encoded outward K ϩ currents in HEK-293 cells were well described by single exponentials at all test potentials. In addition, as reported previously (14,20), the inactivation time constants for the Kv4.x-encoded K ϩ currents do not display any appreciable voltage dependence. The mean Ϯ S.E. decay time constants ( decay ) reported here (Table I) were determined from fits of the decay phases of the currents evoked at ϩ40 mV. To determine the kinetics of recovery (from steady-state inactivation) of the Kv4-encoded K ϩ currents, the pulse protocol described above was used, and the amplitudes of peak outward K ϩ currents evoked during the test pulses (in each cell) were measured and normalized to the amplitude of the peak current determined during the conditioning pulse (in the same cell). Data obtained from several cells were pooled, and mean Ϯ S.E. normalized recovery data were plotted against the interpulse interval and fitted with single exponential functions.
The voltage dependence of steady-state inactivation of the Kv4-encoded K ϩ currents were obtained by normalizing the peak current amplitudes determined for the currents evoked at ϩ50 mV from each conditioning potential to the maximal amplitude of current evoked from the conditioning prepulse of Ϫ110 mV (determined in the same cell).
Data from several cells were pooled, and the mean Ϯ S.E. normalized data were plotted as a function of the prepulse potential and fitted with a Boltzmann equation: at which 50% of the channels are inactivated, and k is a slope factor. All data are presented as means Ϯ S.E. The statistical significance of observed differences between groups of cells was evaluated using a one-way analysis of variance followed by Student-Newman-Keuls test; p values are presented in the text, and statistical significance was set at the p Ͻ 0.05 level.

Expression of NCS-1 in Adult Mouse Ventricular Myocytes-
Western blot analysis of fractionated adult mouse brain and ventricular lysates probed with the anti-NCS-1 antibody revealed the presence of a single Ϸ25-kDa band in both the brain (B) and the ventricular (V) samples (Fig. 1A). Importantly, robust expression of NCS-1 was also evident in Western blots of lysates from isolated mouse ventricular myocytes (M) (Fig. 1A), indicating that the expression of NCS-1 evident in ventricular lysates (V) arises predominantly, if not exclusively, from cardiac myocytes, rather than from non-myocytes, such as intracardiac neurons. In addition, Western blots of lysates prepared from the right ventricle (RV), the left ventricular apex (A), and the ventricular septum (S) reveal that NCS-1 is expressed at similar levels throughout the ventricles of the adult mouse myocardium (Fig. 1B).
Association of NCS-1 with Kv4.3 in Adult Mouse Ventricle-The KChIPs are members of the recoverin-neuronal calcium sensor superfamily, which also includes frequenin, NCS-1, recoverin, calmodulin, neurocalcin, and hippocalcin (21). Recent studies (14,20) have demonstrated that the KChIPs associate with Kv4.x ␣-subunits and modify the gating of Kv4.x-encoded K ϩ channels in heterologous expression systems. It has also been shown that the KChIPs co-immunoprecipate with Kv4.x ␣-subunits from adult rat and mouse brain (14,20) and from adult mouse ventricles (20). To explore the possibility that NCS-1 is also associated with Kv4.x ␣-subunits in (mouse) cardiomyocytes in situ, adult mouse ventricular proteins were immunoprecipitated with a polyclonal anti-Kv4.3 antibody, Kv4.3b, shown previously (20) to be specific for Kv4.3. The resulting immunoprecipitates were then fractionated and probed with another anti-Kv4.3-specific antibody anti-Kv4.3a (20) (Fig. 1C) or with an anti-NCS-1 antibody (Fig. 1D). As reported previously (20), the anti-Kv4.3b antibody reliably precipitates Kv4.3 (IP, Fig. 1C) or Kv4.2 (20) but not Kv ␣-subunits (such as Kv2.1 or Kv1.4) in other subfamilies. When the fractionated immunoprecipitated proteins were immunoblotted with the polyclonal anti-NCS-1 antibody, a single band at Ϸ25 kDa was detected (IP, Fig. 1D), demonstrating that NCS-1 and Kv4.3 are associated in the adult mouse ventricle. The lanes labeled IN (for input) in C and D of Fig. 1 are the lysates prior to immunoprecipitation. The difference in the intensities of the Ϸ25-kDa bands detected with the anti-NCS-1 antibody in the original tissue lysates (IN) and in the samples following immunoprecipitation (IP) with the anti-Kv4.3b antibody (Fig. 1D) suggests that only a fraction of the total NCS-1 protein expressed in adult mouse ventricles is associated with Kv4.3encoded K ϩ channels in vivo (see "Discussion").
Analysis of the decay phases of the currents in records such as those presented in Fig. 2A revealed that the time constants of inactivation ( decay ) of the Kv4.x-encoded K ϩ currents were significantly (p Ͻ 0.01) altered by NCS-1 co-expression. At ϩ40 mV, for example, the mean Ϯ S.E. decay for the Kv4.2-encoded K ϩ currents in the absence and in the presence of NCS-1 are 46 Ϯ 4 and 64 Ϯ 3 ms, respectively (Table I). Co-expression of NCS-1 with Kv4.3 also resulted in marked slowing of the time course of current decay (Table I). In addition, and as reported previously (14), co-expression of KChIP2 significantly (p Ͻ 0.05) slowed the rates of inactivation of the Kv4.2-and the Kv4.3-encoded K ϩ currents (Table I).
By using a double pulse protocol (see "Experimental Procedures"), the effects of NCS-1 and KChIP2 co-expression on the kinetics of recovery from steady-state inactivation of the Kv4.xencoded K ϩ currents were examined. Recordings from representative Kv4.2-(left) and Kv4.3-expressing cells (right) are shown in Fig. 3, and similar results were obtained on many cells ( Fig. 4A and Table I (Table I). In addition, for both Kv4.2 and Kv4.3, the rates of recovery of the currents were accelerated at Ϫ90 mV (Table I).
Co-expression of NCS-1 did not appear to affect the rate of recovery from inactivation of either the Kv4.2-or Kv4.3-encoded K ϩ currents (Fig. 3, middle panels). Analysis of the mean normalized recovery data confirmed that there was no significant difference in the kinetics of recovery of the Kv4.2- (Fig. 4A) or the Kv4.3-encoded K ϩ currents in the presence of NCS-1 ( Table I). The mean Ϯ S.E. recovery time constants for the Kv4.2-and Kv4.3-encoded K ϩ currents at both Ϫ70 and Ϫ90 mV in the presence and absence of NCS-1 were indistinguishable (Table I). In contrast, and as reported previously (14), co-expression of KChIP2 dramatically accelerated the time course of recovery from inactivation of the Kv4.2-and the Kv4.3-encoded K ϩ currents (Fig. 3, bottom panels). The mean Ϯ S.E. time constant of recovery from inactivation of the Kv4.2encoded K ϩ currents at Ϫ70 mV (Fig. 4A) in the presence of KChIP2 (69 Ϯ 5 ms) was significantly (p Ͻ 0.01) faster than in cells expressing Kv4.2 alone (260 Ϯ 21 ms) ( Table I). Coexpression of KChIP2 also markedly accelerated the rate of recovery of the Kv4.2-encoded K ϩ currents at Ϫ90 mV (Table I).
The rates of recovery of the Kv4.3-encoded K ϩ currents expressed in HEK-293 cells were also increased by KChIP2 coexpression (Table I).
As reported previously (14), co-expression of KChIP2 also affected the voltage dependence of steady-state inactivation of heterologously expressed Kv4.x-encoded K ϩ currents. Coexpression of KChIP2 with Kv4.2, for example, resulted in a significant (p Ͻ 0.01) depolarizing shift in the V1 ⁄2 value and increased the steepness (k value) of the normalized current versus conditioning voltage plot (Fig. 4B). Similar results were obtained with Kv4.3 (Table I). Co-expression of NCS-1 also resulted in a (6 mV) depolarizing shift in the voltage dependence of steady-state inactivation of Kv4.3-encoded K ϩ currents, although the slope factor was unaffected (Table I). In contrast, NCS-1 co-expression did not measurably affect the V1 ⁄2 or the k value for the voltage dependence of steady-state inactivation of Kv4.2-encoded K ϩ currents (Fig. 4B). Taken together, these results suggest that NCS-1 modulates the time-and voltagedependent properties of Kv4.x-encoded K ϩ currents, although the effects are less pronounced than seen with KChIP2 (see "Discussion").
In parallel experiments, the effects of decreased NCS-1 expression on the distribution of Kv4.3 in HEK-293 cells were examined. In these experiments, NCS-1 levels in Kv4.3-expressing HEK-293 cells were reduced using an antisense oligonucleotide (AsODN; 2 M) targeted against human NCS-1. As   (Fig. 5B, right panel, Ϫ lanes) was reduced in cells exposed to the NCS-1-AsODN (Fig. 5B, right  panel, ϩ lanes). Control experiments revealed that a randomized control AsODN did not affect Kv4.3 expression (data not shown).

NCS-1 Is Expressed in Adult Mouse
Ventricles and Is Associated with Kv4 ␣-Subunits-The results of these experiments suggest that NCS-1 plays a functional role in the generation of cardiac I to,f channels. Western blot analyses revealed that the anti-NCS-1 antibody identified a single protein of Ϸ25 kDa in adult mouse ventricles as well as in adult mouse brain. The apparent molecular mass (Ϸ25 kDa) of the NCS-1 protein detected here was consistent with predictions based on sequence analysis (21, 31) and another recent report (32) demonstrating robust expression of (22 kDa) NCS-1 in mouse brain. Importantly, although NCS-1 and other members of the recoverinneuronal calcium sensor superfamily are highly expressed in neurons and often considered nervous system-specific proteins (21,31), the results here demonstrated that NCS-1 was also readily detected in extracts of isolated adult mouse ventricular cells. This finding suggested that the NCS-1 evident in the Western blots of lysates of whole adult mouse ventricles reflects predominantly, if not exclusively, expression in myocytes. Therefore, NCS-1 is similar to another distantly related member of the recoverin-neuronal calcium sensor superfamily, KChIP2 (14), which is also highly expressed in adult mouse ventricles (20).
As reported recently by Nakamura and colleagues (32), NCS-1 co-immunoprecipitates with Kv4 ␣-subunits from adult mouse brain. The experiments here revealed that NCS-1 also co-immunoprecipitates from adult mouse ventricles with a specific anti-Kv4.3 antibody, demonstrating directly that NCS-1 associates with Kv4 ␣-subunits in the (adult mouse) myocardium. It has been reported recently (20) that Kv4.2, Kv4.3, and KChIP2 co-immunoprecipitate from adult mouse ventricles, leading to the hypothesis that functional mouse ventricular I to, f   FIG. 3. Co-expression of NCS-1, unlike KChIP2, did not measurably affect the rate of recovery from steadystate inactivation of Kv4.x-encoded K ؉ currents. Outward K ϩ currents were recorded from HEK-293 cells expressing Kv4.2 (left) or Kv4.3 (right) alone (top) or in combination with NCS-1 (1:1) (middle) or KChIP2 (1:1) (bottom). To examine the rates of recovery from inactivation, the Kv4.x-encoded currents were first inactivated during 400-ms conditioning pulses to ϩ50 mV. Cells were then hyperpolarized to Ϫ70 mV for varying times (ranging from 10 ms to 2 s) before 400-ms test depolarizations to ϩ50 mV were presented to assess the extent of recovery. In contrast to the effects of KChIP2 (bottom), co-expression of NCS-1 (middle) did not measurably affect the rates of recovery of the Kv4.2-or the Kv4.3-induced K ϩ currents (see also Fig. 4 and Table I). Peak outward K ϩ currents following each recovery period were measured and normalized to the peak current amplitudes measured after the 10-s recovery period (in the same cell). Mean Ϯ S.E. normalized recovery data were then determined for the currents produced on expression of Kv4.2 alone (E) or Kv4.2 in combination with NCS-1 (q) or KChIP2 (Ⅺ). B, the voltage dependence of steady-state inactivation of the Kv4.2-encoded K ϩ currents were determined using the standard two pulse protocol described under "Experimental Procedures." The currents evoked from each conditioning potential were measured and normalized to the current evoked from Ϫ110 mV (in the same cell). Mean Ϯ S.E. normalized current amplitudes were plotted as a function of conditioning potential for the Kv4.2-encoded K ϩ currents (E) and for the currents produced on co-expression in Kv4.2 with KChIP2 (Ⅺ) or NCS-1 (q). The solid lines represent the best (single) Boltzmann fits to the mean Ϯ S.E. normalized data. channels are heteromeric consisting of both Kv4 ␣-subunits and KChIP2. The immunoprecipitation results here suggest that NCS-1 also contributes to the formation of heteromeric mouse ventricular I to,f channels. Importantly, however, and in contrast to the findings with KChIP2 (20), only a fraction of the total ventricular NCS-1 protein co-immunoprecipitated with Kv4.3. The simplest interpretation of this finding is that NCS-1 likely subserves functional roles in mouse ventricular myocytes in addition to contributing to the formation of I to,f channels. This hypothesis is consistent with previous studies (21,(33)(34)(35)(36) in neurons suggesting that NCS-1 is a multifunctional protein.
Clearly, additional experiments focused on testing this hypothesis and exploring the functional roles of NCS-1 in the mammalian myocardium are warranted.
The Western blot data presented reveal that NCS-1 is expressed at similar levels in adult mouse right ventricle, left ventricular apex, and septum, i.e. there was no apparent "gradient" of NCS-1 protein expression in adult mouse ventricles. These observations are similar to those reported recently (20) for KChIP2 in adult mouse ventricles in that KChIP2 protein expression levels in right ventricles, left ventricular apex, and septum are also indistinguishable. It has also been demonstrated that the KChIP2 message is uniformly expressed in adult rat ventricles (37). In the ventricles of larger mammals, such as canine and human, however, KChIP2 mRNA expression reportedly varies through the thickness of the ventricular wall (37). These observations, together with the finding that Kv4.3 message levels are similar throughout the ventricles, have been interpreted as suggesting that the differential expression of KChIP2 underlies the transmural gradient in I to,f density in human and canine ventricles (37). Nevertheless, it will be of interest to examine NCS-1 (message and protein) expression in canine and human myocardium. In mouse and rat ventricles, the uniform expression of KChIP2 (20, 37) and NCS-1 (present study) contrasts with the heterogeneous expression of Kv4.2 mRNA (6,38) and protein (20,38) and the regional differences in I to,f density in these animals (38,39). Indeed, in rats and mice, it appears that Kv4.2, not the Kv accessory subunits, is the primary determinant of the gradient in ventricular I to,f expression (6,20,38).
NCS-1 Regulates the Membrane Expression and the Properties of Kv4 ␣-Subunits/Channels-Similar to KChIP2 (14,20), co-expression of NCS-1 with Kv4 ␣-subunits in HEK-293 cells significantly increased K ϩ current densities and slowed inactivation. Similar results have been reported recently (32) for NCS-1 co-expression with Kv4.2 or Kv4.3 in Xenopus oocytes. In contrast to the effects of KChIP2 (14,20), however, coexpression of NCS-1 had little or no effect on the voltage dependence of steady-state inactivation or the kinetics of recovery from inactivation of Kv4.x-encoded K ϩ currents in HEK-293 cells. Interestingly, co-expression of NCS-1 reportedly does result in measurable acceleration of the rates of recovery from inactivation of Kv4.2-and Kv4.3-encoded K ϩ currents in Xenopus oocytes (32).
The biochemical data presented here demonstrate that NCS-1 overexpression (in HEK-293 cells) increases the membrane expression of Kv4.3 and, conversely, that reducing NCS-1 attenuates the membrane expression of Kv4.3. In addition, the immunohistochemical experiments revealed that the overexpression of NCS-1 increased the cell surface expression of Kv4.3. Thus, it seems reasonable to suggest that NSC-1 forms complexes with Kv4.x ␣-subunits and functions to increase the cell surface expression of Kv4.x proteins and the densities of Kv4.x-encoded K ϩ channels. These results clearly highlight the complexity of the molecular basis of cardiac I to,f channels, which appear to reflect heteromeric assembly of poreforming Kv4.x ␣-subunits and diverse accessory subunits, including KChIP2 and NCS-1. Further studies aimed at defining the sites of interactions between Kv4.x ␣-subunits and NCS-1, determining the involvement of other accessory subunits and/or regulatory molecules in mediating these interactions, and delineating the underlying molecular mechanisms involved are warranted.
Relationship to Previous Studies-The widespread distribution of NCS-1 in the mammalian brain and spinal cord (21,31,33) and the subcellular localization of NCS-1 (33) suggest that the NCS-1 protein functions in the regulation of a variety of neuronal target proteins, including ion channels. In addition, the fact that NCS-1 is abundantly expressed in the Golgi apparatus and in neurofilament-rich structures has been interpreted as suggesting that NCS-1 likely functions in protein trafficking and cytoskeletal interactions (33). Although it has Approximately 48 h later, the cultures were fixed (see "Experimental Procedures") and probed with a specific polyclonal anti-Kv4.2 antibody, followed by a Cy3-conjugated goat-anti-rabbit secondary antibody. In cells expressing Kv4.2 alone (A), the anti-Kv4.2 antibody labeling is diffuse, whereas in cells co-expressing NCS-1 (B), the labeling is punctate and appears predominantly at the cell surface (arrowheads). long been recognized that NCS-1 plays a role in the regulation of synaptic transmission (34), direct support for a role for NCS-1 in the functioning of membrane ion channels was provided only recently (35) with the demonstration that NCS-1 regulates voltage-gated Ca 2ϩ channel currents in adrenal chromaffin cells. In addition, it has been reported that the effects of glial derived neurotrophic factor (GDNF) on motoneurons are mediated by NCS-1 (36), suggesting an important role for NCS-1 in the functioning of neuronal Ca 2ϩ channels. In support of this hypothesis, it was recently reported (40) that NCS-1 directly modulates the activity of P/Q-type voltage-gated Ca 2ϩ currents in presynaptic nerve terminals.
The recent report by Nakamura and colleagues (32) and the results presented here clearly also reveal interactions between NCS-1 and the Kv4.x ␣-subunit-encoded fast transient outward K ϩ currents, referred to as I A and I to,f , in neurons (32) and myocardial cells (1,20,39), respectively. Interestingly, it has also been reported (41) that I A in midbrain dopaminergic neurons is regulated by GDNF, through a mitogen-activated protein kinase-dependent pathway. Given the regulatory role of NCS-1 in the modulation of voltage-gated Ca 2ϩ channels by GDNF (36), it seems reasonable to suggest that the effects of GDNF on I A may also be mediated by NCS-1. Further experiments focused on testing this hypothesis directly, as well as on exploring the possibility that NCS-1 plays a role in the regulation/modulation of other voltage-gated K ϩ channels in neurons and cardiac cells, will clearly be of interest.