Role of Heteromultimers in the Generation of Myocardial Transient Outward K+ Currents
Previous studies have demonstrated a role for Kv4 α subunits in the generation of the fast transient outward K+ current, Ito,f, in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular Ito,f. Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in Ito,f density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3 selectively attenuate Ito,f in mouse ventricular cells. Adenoviral-mediated coexpression of Kv4.2 and Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces transient outward K+ currents with properties distinct from those produced on expression of Kv4.2 or Kv4.3 alone, and the gating properties of the heteromeric Kv4.2/Kv4.3 channels in ventricular cells are more similar to native Ito,f than are the homomeric Kv4.2 or Kv4.3 channels. Biochemical studies reveal that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from adult mouse ventricles. In addition, most of the Kv4.2 and KChIP2 are associated with Kv4.3 in situ. Taken together, these results demonstrate that functional mouse ventricular Ito,f channels are heteromeric, comprising Kv4.2/Kv4.3 α subunits and KChIP2. The results here also suggest that Kv4.2 is the primary determinant of the regional heterogeneity in Ito,f expression in adult mouse ventricle.
Two distinct transient outward K+ currents, the rapidly inactivating and recovering Ito,fast (Ito,f) and the slowly inactivating and recovering Ito,slow (Ito,s), have been distinguished in the mammalian myocardium, and these two K+ currents are differentially distributed in human, ferret, rabbit, rat, and mouse hearts.1–5⇓⇓⇓⇓ In ventricular myocytes isolated from mice with a targeted deletion of the Kv1.4 gene, Ito,s is selectively eliminated, demonstrating directly that the Kv1.4 α subunit underlies Ito,s.6 Considerable evidence has also accumulated documenting a role for Kv4 α subunits in the generation of cardiac Ito,f. Dixon and McKinnon,7 for example, reported that the Kv4.2 message level varies through the thickness of the ventricular wall in rat, in parallel with regional differences in Ito,f. In addition, Ito,f in rat ventricular cells is attenuated on exposure to antisense oligodeoxynucleotides targeted against either Kv4.2 or Kv4.3,8 as well as after exposure to an adenoviral construct encoding a truncated Kv4.2 subunit.9
Subsequently, Barry et al10 reported that Ito,f is eliminated in ventricular cells isolated from transgenic mice expressing a pore mutant of Kv4.2, Kv4.2W362F, that functions as a dominant-negative. Because functional voltage-gated K+ channels comprise 4 pore-forming α subunits11 and both Kv4.2 and Kv4.3 are expressed in rodent myocardium, it is presently unclear whether functional Ito,f channels reflect the homomultimeric assembly of Kv4.2 and Kv4.3 or the presence of heteromultimeric Kv4.2/Kv4.3 channels. Coexpression of Kv4.2W362F with wild-type Kv4.3 in QT-6 cells reveals no functional K+ currents, demonstrating that Kv4.2 and Kv4.3 coassemble in vitro.10 Previous studies have not, however, examined the biophysical properties of coexpressed Kv4.2+Kv4.3-encoded K+ channels or explored the possibility that these subunits are associated in rodent myocardium in vivo. In addition, accessory subunits, such as the Kv channel interacting proteins (KChIPs), modulate the expression and properties of Kv4-encoded K+ channels.12,13⇓ In heterologous systems, coexpression of KChIP with Kv4 α subunits markedly increases current densities and accelerates recovery from inactivation.12 The observation that KChIP2 is expressed in rodent heart12,14⇓ suggests that this subunit may assemble with Kv4 α subunits to produce Ito,f.
In the experiments here, the currents produced on expression of Kv4.2, Kv4.3, or Kv4.2+Kv4.3 in HEK-293 cells and in mouse ventricular myocytes were examined and compared with endogenous mouse ventricular Ito,f. The results reveal that the time- and voltage-dependent properties of heteromeric Kv4.2/Kv4.3 K+ channels are quantitatively similar to those of endogenous Ito,f and distinct from homomeric Kv4.2- or Kv4.3-encoded currents. Biochemical studies demonstrate that Kv4.3 and KChIP2 are uniformly expressed, whereas Kv4.2 expression varies, and that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from mouse ventricle. Taken together, these results reveal that mouse ventricular Ito,f channels are generated by heteromeric assembly of Kv4.2/Kv4.3 and KChIP2 and that the differential expression of Kv4.2 underlies the regional heterogeneity in Ito,f density in the mouse heart.
Materials and Methods
Animals used in the present study were handled in accordance with guidelines published in the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health).
An expanded online Materials and Methods section containing details of cell culture, antisense experiments, adenoviral gene transfer, Western blots, immunoprecipitations, and electrophysiological recordings is available in the online data supplement at http://www.circresaha.org.
Expression of Kv4.2 and Kv4.3 in Adult Mouse Ventricles
Outward K+ current waveforms in cells isolated from adult mouse right ventricle (RV), left ventricular apex (LVA), and septum (LVS) are distinct.5,6,15⇓⇓ These differences reflect variations in the densities of the transient outward currents Ito,f and Ito,s, whereas no regional differences in IK,slow or ISS are evident.5,6,15⇓⇓ Previous studies have also shown that both Kv4.2 and Kv4.3 are expressed at the mRNA level in adult mouse ventricles.16 Initial experiments here, therefore, were focused on examining the patterns and levels of expression of Kv4.2 and Kv4.3 proteins using subunit-specific antibodies (see online data supplement at http://www.circresaha.org). As illustrated in Figure 1, a single band at ≈70 kDa is detected with the anti-Kv4.2a antibody (Figure 1A), and a single band at ≈76 kDa is evident using the anti-Kv4.3a antibody (Figure 1B). Both bands were eliminated when the antibodies were incubated with the peptides against which each was generated (not illustrated). The intensity of the band detected with the anti-Kv4.3a antibody is similar in RV, LVA, and LVS, whereas Kv4.2 protein expression is higher in RV and LVA than in LVS. The differences in Kv4.2 expression, therefore, parallel the regional heterogeneity in Ito,f density in adult mouse ventricles.5,6,15⇓⇓
Effects of AsODN-Kv4.2 and AsODN-Kv4.3 on Mouse Ventricular K+ Currents
The effects of antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 and Kv4.3 on the outward K+ currents in mouse ventricular cells were also examined. Representative current waveforms recorded from cells exposed to 1 μmol/L randomized control AsODN, AsODN-Kv4.2, AsODN-Kv4.3, or to both AsODN-Kv4.2 and AsODN-Kv4.3 (1 μmol/L each) are illustrated in Figure 2A. The densities and the waveforms of the currents recorded from cells treated with the randomized control AsODN (n=49) are not significantly different from those in untreated cells (n=21) (data not shown). Peak outward K+ currents are markedly reduced, however, in cells exposed to AsODN-Kv4.2 (n=23), AsODN-Kv4.3 (n=16), or to both AsODNs (n=18) (Figure 2B). The densities of the K+ currents remaining at the end of the depolarizing voltage steps in AsODN-treated cells, in contrast, are indistinguishable from controls (Figure 2B).
Previous studies have documented the expression of 4 components of the outward K+ currents in adult mouse ventricular myocytes Ito,f, Ito,s, IK,slow, and Iss.5 Although initially distinguished based on differences in kinetic properties and pharmacological sensitivities,5 it has now been clearly demonstrated that these currents reflect the expression of distinct molecular entities.6,10,15,17,18⇓⇓⇓⇓ It has been shown, for example, that Kv4α subunits encode Ito,f,10,15⇓ whereas Kv1.4 underlies Ito,s.6,15⇓ In addition, there are 2 components of mouse ventricular IK,slow, one encoded by Kv1.517 and the other by Kv2 α subunits.18 In addition to identifying the Kv α subunits underlying mouse ventricular K+ channels, these findings validate the kinetic analyses of the decay phases of the outward K+ currents to quantify the expression of Ito,f, Ito,s, IK,slow, and Iss. In the present experiments, the decay phases of the currents were analyzed (see online data supplement) to obtain the amplitudes (densities) of Ito,f, Ito,s, IK,slow, and Iss. These analyses revealed that exposure to either AsODN-Kv4.2 or AsODN-Kv4.3 selectively attenuates Ito,f by ≈35% and ≈55%, respectively (Figure 2B). In contrast, the densities of IK,slow and Iss5 are unaffected. Interestingly, exposure of cells (n=18) to both AsODN-Kv4.2 and AsODN-Kv4.3 did not produce further attenuation of Ito,f (Figure 2B). In addition, the τdecay values for Ito,f are not affected in cells exposed to AsODNs (Figure 2C).
Homomeric and Heteromeric Kv4 α Subunit–Encoded K+ Currents
Although the antisense experiments described above reveal that Kv4.2 and Kv4.3 contribute to mouse ventricular Ito,f, it remains unclear whether functional Ito,f channels reflect the homomeric assembly of Kv4.2 and Kv4.3 or heteromeric assembly of both Kv4 α subunits. To compare the biophysical properties of homomeric Kv4.2 or Kv4.3 and heteromeric Kv4.2/Kv4.3 channels, Kv4.2, Kv4.3, or Kv4.2+Kv4.3 (in a ratio of 1:1) and EGFP were introduced into HEK-293 cells using inducible adenoviral gene transfer, and whole-cell recordings were obtained from EGFP-positive cells 20 to 24 hours after induction (see online data supplement). As is evident in Figure 3A, coexpression of Kv4.2 and Kv4.3 reveals rapidly activating and inactivating K+ currents. Mean±SEM peak outward K+ current densities (at +40 mV) in cells expressing Kv4.2 (51±5 pA/pF, n=19), Kv4.3 (60±9 pA/pF, n=13), or both Kv4.2 and Kv4.3 (56±7 pA/pF, n=12) are similar, demonstrating that coexpression of Kv4.2 and Kv4.3 does not increase functional cell surface channel density. In addition, the decay phases of the Kv4.2+Kv4.3 currents are well described by single exponentials with a mean±SEM τdecay value intermediate between the values determined for the currents produced on expression of Kv4.2 or Kv4.3 alone (Table).
The kinetics of recovery from steady-state inactivation of the currents produced on coexpression of Kv4.2 and Kv4.3 are also distinct. At −70 mV, the Kv4.2+Kv4.3-induced currents recover more rapidly than the currents produced on expression of Kv4.2 or Kv4.3 alone (Figure 3B). At more negative potentials (−90 mV), the recovery rates for the Kv4.2+Kv4.3-, as well as the Kv4.2- and Kv4.3-, encoded currents are accelerated (Table). At both −70 and −90 mV, however, recovery of the Kv4.2+Kv4.3-induced currents is monoexponential, with mean±SEM recovery time constants significantly (P<0.01) faster than those determined for either the Kv4.2- or the Kv4.3-induced currents (Table). In addition, there are differences in the voltage dependences of steady-state inactivation of the currents (Figure 5A). The V1/2 for steady-state inactivation of the Kv4.2+Kv4.3-induced currents is depolarized relative to the V1/2 for the Kv4.2- and Kv4.3-encoded currents (Table).
Heteromeric Kv4.2/Kv4.3 Channels in Mouse Ventricular Cells
The results obtained in HEK-293 cells suggest that coexpression of Kv4.2 and Kv4.3 results in the preferential formation of heteromeric K+ channels with properties distinct from homomeric Kv4.x channels. To examine the properties of homomeric and heteromeric Kv4.x-encoded K+ channels in cardiomyocytes, Kv4.2, Kv4.3, and EGFP were introduced into isolated mouse ventricular cells using adenoviral gene transfer. No differences in outward K+ current densities or properties in AdEGI-infected (n=16) and uninfected (n=10) cells examined 48 hours after plating were evident (data not shown), suggesting that expression of EGFP alone does not influence K+ currents in these cells. Peak outward K+ currents in AdEGI-Kv4.2-, AdEGI-Kv4.3-, and AdEGI-Kv4.2+AdEGI-Kv4.3-infected cells, however, are larger than the currents in AdEGI-infected cells (Figure 4A). Mean±SEM densities (at +40 mV) of the rapidly inactivating current component in Kv4.2- (20±2 pA/pF, n=16), Kv4.3- (22±3 pA/pF, n=15), and Kv4.2+Kv4.3- (24±3 pA/pF, n=18) expressing cells are significantly (P<0.001) higher than in AdEGI-infected cells (9±1 pA/pF, n=16). Although inactivation of the Kv4.3-induced currents is slow, the mean±SEM τdecay values for the Kv4.2- and Kv4.2+Kv4.3-encoded currents and endogenous Ito,f are similar (Table).
Coexpression of Kv4.2 and Kv4.3 results in a marked depolarizing shift in the voltage dependence of steady-state inactivation compared with the currents produced on expression of Kv4.2 or Kv4.3 alone (Figure 5B). In addition, the steady-state inactivation curve for the Kv4.2+Kv4.3-encoded currents parallels endogenous Ito,f (Figure 5B); both the slope factor (k) and the V1/2 are very similar to Ito,f (Table). The Kv4.2- and the Kv4.3-induced K+ currents in mouse ventricular cells recover slowly from steady-state inactivation, whereas coexpression of Kv4.2 and Kv4.3 produces currents with markedly accelerated recovery (Figure 4B). Although significantly (P<0.001) faster than the Kv4.2- or the Kv4.3-encoded currents (Table), the rates of recovery of the heteromeric Kv4.2+Kv4.3 currents are significantly (P<0.01) slower than endogenous Ito,f (see Discussion).
Association of Kv4.2 and Kv4.3 In Vivo
The results of the experiments described suggest that endogenous mouse ventricular Ito,f channels reflect heteromeric assembly of Kv4.2 and Kv4.3. To determine if Kv4.2 and Kv4.3 indeed associate in vivo, immunoprecipitation experiments were performed using anti-Kv4 subunit specific antibodies. Membrane fractions were immunoprecipitated with the anti-Kv4.2a antibody and 2 different anti-Kv4.3 antibodies, anti-Kv4.3a and anti-Kv4.3b (see online data supplement). Although all 3 antibodies immunoprecipitate Kv4.2 and/or Kv4.3 from mouse brain (Figures 6A through 6C), only the anti-Kv4.3b antibody precipitates Kv4.3 from mouse ventricular membrane preparations (Figure 6C).
To determine if Kv4.2 associates with Kv4.3, the immunoprecipitates obtained with the anti-Kv4.3b antibody were resolved by SDS-PAGE, transferred to PVDF membranes, and blotted with the anti-Kv4.2a antibody. A band at ≈70 kDa was detected in mouse brain and ventricles, demonstrating that Kv4.2 coimmunoprecipitates with Kv4.3 (Figure 6D). In addition, only a small fraction of the total Kv4.2 remains in the supernatant after immunoprecipitation with the anti-Kv4.3b antibody (Figure 6E), indicating that most of Kv4.2 protein is associated with Kv4.3 in adult mouse ventricles. In contrast, Kv2.1 is not immunoprecipitated with the anti-Kv4.3b antibody (Figure 6F), although Kv2.1 is readily detected in Western blots of fractionated mouse ventricular membrane proteins.
Heteromeric Assembly of KChIP2 and Kv4 α Subunits in Mouse Heart
Western blots revealed that KChIP2 is also readily detected in adult mouse ventricles (and brains) with a monoclonal anti-KChIP2 antibody (Figure 7A). Similar to previous findings in rat neocortex,12 the monoclonal anti-KChIP2 antibody identifies a single protein band at ≈ 35 kDa in mouse brain and ventricles (Figure 7A). Similar results are obtained with a polyclonal anti-KChIP2 antibody (Figure 7B), and indeed, both antibodies recognize the same protein (Figure 7C). In contrast to the regional heterogeneities in Ito,f density5,6,15⇓⇓ and Kv4.2 protein expression (Figure 1A), however, KChIP2 is expressed at equal levels in adult mouse RV, LVA, and LVS (Figures 7A and 7B).
To determine if KChIP2 associates with Kv4 α subunits in vivo, the immunoprecipitates obtained with the anti-Kv4.3b antibody were resolved and immunoblotted with the monoclonal anti-KChIP2 antibody. A single band at ≈35 kDa was detected with the monoclonal anti-KChIP2 antibody and only a small fraction of the total KChIP2 remains in the supernatant after precipitation (Figure 7E). In addition, Western blots of immunoprecipitates obtained using the monoclonal anti-KChIP2 antibody revealed that both Kv4.2 and Kv4.3 coimmunoprecipitate with KChIP2 from adult mouse ventricles (Figure 7D). In contrast, neither Kv2.1 nor Kv2.2 immunoprecipitates with KChIP2 (not shown), demonstrating that KChIP2 preferentially associates with Kv4 α subunits in adult mouse ventricles.
Heteromultimers of Kv4.2 and Kv4.3 Underlie Mouse Ventricular Ito,f
The results of the experiments here reveal that both Kv4.2 and Kv4.3 contribute to the generation of functional mouse ventricular Ito,f channels. Although both are abundant at the protein level in adult mouse ventricles, the expression patterns of Kv4.2 and Kv4.3 are distinct. Kv4.3 appears to be uniformly expressed in the ventricles, whereas Kv4.2 expression parallels the regional heterogeneity in Ito,f density.5,6,15⇓⇓ The antisense experiments revealed that Ito,f is selectively attenuated in isolated mouse ventricular cells exposed to either AsODN-Kv4.2 or AsODN-Kv4.3. Interestingly, simultaneous exposure to both AsODNs did not produce larger reduction of Ito,f density than observed with either AsODN alone. Although this observation might suggest that mouse ventricular Ito,f channels reflect heteromeric assembly of Kv4.2 and Kv4.3, the possibility of nonlinear AsODN effects3,19⇓ cannot be excluded.
Although expression of Kv4.2 or Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces rapidly activating and inactivating K+ currents, the properties of the currents are distinct from endogenous (mouse) ventricular Ito,f (Table). Coexpression of Kv4.2 and Kv4.3 in HEK-293 cells produces transient outward K+ currents with accelerated recovery kinetics and a depolarizing shift in the voltage dependence of steady-state inactivation compared with Kv4.2- or Kv4.3-encoded currents (Table). These observations suggest that Kv4.2 and Kv4.3 preferentially form heteromeric channels with properties distinct from homomeric Kv4.2 or Kv4.3 channels. The myocyte studies reveal that heteromeric Kv4.2/Kv4.3 channels are similar to mouse ventricular Ito,f. In addition, biochemical evidence is presented demonstrating that Kv4.2 and Kv4.3 coimmunoprecipitate and that most of Kv4.2 protein in adult mouse ventricles is associated with Kv4.3. Taken together, these results are interpreted as revealing that heteromultimers of Kv4.2 and Kv4.3 underlie mouse ventricular Ito,f.
It is well documented that heterologous expression of Kv α subunits in the same subfamily produces heteromeric channels.23 In addition, it has been shown that Kv1 α subunits coimmunoprecipitate from (rat) brain.23 To our knowledge, however, this is the first demonstration of heteromeric assembly of Kv4 α subunits in vivo. Importantly, this study also establishes the functional significance of heteromeric assembly of Kv4.2 and Kv4.3 in the generation of myocardial Ito,f. These results can be contrasted with cardiac Kv1 α subunits that encode distinct K+ channels. Kv1.4, for example, underlies Ito,s,6 whereas Kv1.5 encodes the μmol/L 4-aminopyridine–sensitive component of IK,slow in mouse ventricular cells.17
Relationship to Previous Studies on the Molecular Correlates of Ito,f
In spite of the marked diversity of repolarizing K+ currents, the properties of Ito,f in different cardiac cell types and species are similar, suggesting that the molecular correlates of the underlying K+ channels are also the same.23 Considerable experimental evidence has now been provided documenting a role for Kv4 α subunits, Kv4.2 and/or Kv4.3, in the generation of cardiac Ito,f.23 Previous studies, focused on Kv α subunit mRNA and protein expression levels, as well as the effects of AsODNs, suggested that both Kv4.2 and Kv4.3 contribute to Ito,f in rat ventricular myocytes.7,8,25⇓⇓ The experiments here clearly demonstrate that mouse ventricular Ito,f is generated by heteromultimers of Kv4.2 and Kv4.3. Nevertheless, the fact that Kv4.2 appears not to be expressed in the ventricles of large mammals, such as dog and human,26 clearly suggests that the molecular composition(s) of functional Ito,f channels in these species are not identical to mouse ventricular Ito,f.
It certainly also seems possible that the molecular basis of Ito,f might be different in mouse atria. In rat atrial cells, reductions in Ito,f density are observed after exposure to AsODN-Kv4.2 but not to AsODN-Kv4.3, suggesting that Kv4.2 and Kv4.3 do not assemble in rat atria in vivo and that Kv4.3 does not contribute to rat atrial Ito,f.19 It has also been reported that AsODN-Kv4.2 or AsODN-Kv1.4 attenuates Ito in rabbit atrial myocytes.3 In human atrial cells, however, only AsODN-Kv4.3 affects Ito,f, suggesting that only Kv4.3 contributes to human atrial Ito,f.3 Recently, it was reported that Kv4.1 is expressed (at the message level) in human heart.27 Experiments aiming at determining the roles of homomeric Kv4.x and heteromeric Kv4.2/Kv4.3 and/or Kv4.1/Kv4.3 channels in the generation of Ito,f in other species will clearly be of interest.
Roles of Accessory Subunits in the Generation of Myocardial Ito,f
Although coexpression of Kv4.2 and Kv4.3 in mouse ventricular myocytes produces K+ currents similar to endogenous Ito,f, there are some subtle, but statistically significant, differences (Table). The rate of recovery of the Kv4.2+Kv4.3-encoded currents, for example, is slower than the rate of recovery of the endogenous Ito,f. These observations suggest that there are additional components of endogenous cardiac Ito,f channels. Indeed, a number of Kv channel accessory subunits have been identified and shown to modulate the properties and/or the expression of Kv α subunit–encoded K+ currents in expression systems.12–14,23,28,29⇓⇓⇓⇓⇓ In addition, some of these subunits interact with Kv4 α subunits.12–14,30–32⇓⇓⇓⇓⇓ When coexpressed in HEK-293 cells, for example, Kvβ1.2 confers O2 sensitivity to Kv4.2-induced K+ currents31 and Kvβ2.1 associates with Kv4.3 in rat brain.32
The Kv channel interacting proteins (KChIPs) have been shown to associate with Kv4 α subunits in (rat) brain and to modify the properties and the expression of Kv4.x-encoded K+ currents.12 The results here reveal that KChIP2 preferentially associates with Kv4.2 and Kv4.3 in adult mouse ventricles, suggesting that KChIP2 is likely an integral component of myocardial Ito,f channels. Consistent with this hypothesis, it was recently reported that Ito (Ito,f) is eliminated in ventricular myocytes isolated from mice lacking KChIP2 (KChIP2−/−).33 The complete loss of Ito,f in KChIP2−/− ventricular myocytes is surprising, given that expression of Kv4.x α subunits alone reveals robust voltage-gated K+ currents20–23⇓⇓⇓ and that the properties of these currents are modified substantially by KChIP coexpression.12 In addition, no QT prolongation was reported in KChIP2−/− mice, and these animals display increased susceptibility to ventricular tachycardia.33 These observations contrast with findings in mice in which Ito,f is eliminated by expression of a dominant-negative Kv4.2 construct, Kv4.2W362F,10,15⇓ or deletion of Kv4.2 (Kv4.2−/−).34 In Kv4.2W362F and Kv4.2−/− mice, QT intervals are prolonged and these animals are resistant to arrhythmias,10,15⇓ owing to the reduced dispersion of repolarization resulting from the loss of Ito,f.35 Taken together, these observations suggest a role for KChIP2 in addition to the regulation of Kv4-encoded Ito,f channels. Experiments aimed at testing this hypothesis and further studies on the KChIP2−/− mice will be of interest.
The fact that KChIP2 associates with Kv4.2 and Kv4.3 suggests that it is theoretically possible that mouse ventricular Ito,f could reflect a mixture of homomeric Kv4.2- and Kv4.3-encoded channels (coassembled with KChIP2). This hypothesis seems unlikely, however, because the biochemical data demonstrate that most of the Kv4.2 protein is found in association with Kv4.3 in adult mouse ventricles. In addition, Ito,f is eliminated and no Kv4.3-like currents are detected in Kv4.2−/− ventricular myocytes, despite the fact that Kv4.3 protein expression is not affected by the elimination of Kv4.2.34 These findings clearly support a role for heteromeric Kv4.2/Kv4.3 assembly in the generation of mouse ventricular Ito,f. These results also suggest that accessory subunits modulate the assembly, processing, and/or targeting of heteromeric Kv4.2/Kv4.3-encoded channels. The lack of stoichiometric amounts of accessory subunits may underlie the subtle (but statistically significant) differences in the gating properties of endogenous Ito,f and the currents produced on adenoviral-induced coexpression of Kv4.2 and Kv4.3 in mouse ventricular cells (see Table).
The results of the biochemical studies here also reveal that the expression of Kv4.2, but not Kv4.3 or KChIP2, parallels the regional heterogeneity in Ito,f density in mouse ventricles. These findings are consistent with a recent study demonstrating that there is no gradient of KChIP2 mRNA expression in adult rat ventricles.14 In human and canine ventricles, however, KChIP2 mRNA levels are reportedly higher in left ventricular epicardium than endocardium, whereas Kv4.3 mRNA levels are similar across the left ventricular wall.14 These observations suggest a divergence of molecular mechanisms regulating Ito,f expression in small versus large mammals. Clearly, further experiments aimed at exploring the detailed mechanisms underlying myocardial Ito,f expression are warranted.
The authors gratefully acknowledge the financial support provided by the McDonnell Center for Cellular and Molecular Neurobiology (fellowship to W.G.), the NIH (NS42225 to J.S.T. and HL34161 to J.M.N.), the CARE Foundation, and the Blaustein Pain Foundation (Research Career Development Awards to D.C.J). The authors also thank Lynne Buchwalder and Karen Carroll for technical assistance in generating and characterizing the anti-KChIP antibodies.
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received April 30, 2001; resubmission received September 19, 2001; revised resubmission received January 30, 2002; accepted January 30, 2002.
- ↵Näbauer M, Beuckelmann DJ, Uberführ P, Steinbeck G. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation. 1996; 93: 168–177.
- ↵Brahmajothi MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne JM, Strauss HC. Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol. 1999; 113: 581–600.
- ↵Wang Z, Feng J, Shi H, Pond A, Nerbonne JM, Nattel S. Potential molecular basis of different physiological properties of the transient outward K+ current in rabbit and human atrial myocytes. Circ Res. 1999; 84: 551–561.
- ↵Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated outward K+ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661–678.
- ↵Dixon JE, McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res. 1994; 75: 252–260.
- ↵Johns DC, Nuss HB, Marban E. Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs. J Biol Chem. 1997; 272: 31598–31603.
- ↵Barry DM, Xu H, Schuessler RB, Nerbonne JM. Functional knockout of the transient outward current, long QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 α subunit. Circ Res. 1998; 83: 560–567.
- ↵Holmqvist MH, Cao J, Knoppers MH, Jurman ME, Distefano PS, Rhodes KJ, Xie Y, An WF. Kinetic modulation of Kv4-mediated A-current by arachidonic acid is dependent on potassium channel interacting proteins. J Neurosci. 2001; 21: 4154–4161.
- ↵Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of Ito,f and Ito,s: early afterdepolarizations, atrioventricular block and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 α subunit. Circ Res. 2000; 87: 73–79.
- ↵London B, Guo W, Pan X-H, Lee JS, Shusterman V, Rocco CJ, Logothetis DA, Nerbonne JM, Hill JA. Targeted replacement of Kv1.5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of IK,slow and resistance to drug-induced QT prolongation. Circ Res. 2001; 88: 940–946.
- ↵Xu H, Barry DM, Li H, Brunet S, Guo W, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant negative Kv2 α subunit. Circ Res. 1999; 85: 623–633.
- ↵Franqueza L, Valenzuela C, Eck J, Tamkun MM, Tamargo J, Snyders DJ. Functional expression of an inactivating potassium channel (Kv4.3) in a mammalian cell line. Cardiovasc Res. 1999; 41: 212–219.
- ↵Yeola SW, Snyders DJ. Electrophysiological and pharmacological correspondence between Kv4.2 current and cardiac transient outward current. Cardiovasc Res. 1997; 33: 540–547.
- ↵Trimmer JS, Rhodes KJ. Heteromultimer formation in native K+ channels.In: Archer SL, Rusch NJ, eds. Potassium Channels in Cardiovascular Biology. New York, NY: Kluwer Academic/Plenum Publisher; 2001: 163–176.
- ↵Barry DM, Trimmer JS, Merlie JP, Nerbonne JM. Differential expression of voltage-gated K+ channel subunits in adult rat heart: relation to functional K+ channels. Circ Res. 1995; 77: 361–369.
- ↵Dixon JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, McKinnon D. Role of the Kv4.3 K+ channel in ventricular muscle: a molecular correlate for the transient outward current. Circ Res. 1996; 79: 659–668.
- ↵Nakahira K, Shi G, Rhodes KJ, Trimmer JS. Selective interaction of voltage-gated K+ channel β-subunits with alpha-subunits. J Biol Chem. 1996; 271: 7084–7089.
- ↵Pérez-García MT, López-López JR, González C. Kvβ1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to Kv4.2 but not to Shaker channels. J Gen Physiol. 1999; 113: 897–907.
- ↵Yang E-K, Alvira MR, Levitan ES, Takimoto K. Kvβ subunits increase expression of Kv4.3 channels by interacting with their C termini. J Biol Chem. 2001; 276: 4839–4844.
- ↵Kuo H-C, Cheng C-F, Clark RB, Lin JJ-C, Hoshijima M, Nguyen-Tran VTB, Gu Y, Ikeda Y, Chu P-H, Ross J Jr, Giles WR, Chien KB. A defect in the Kv channel interacting protein 2 (KChIP2) gene leads to a complete loss of Ito and confers susceptibility to ventricular tachycardia. Cell. 2001; 107: 801–813.
- ↵Baker LC, Guo W, Nerbonne JM, Coi B-R, Salama G. A decrease in dispersion of repolarization (DR) protects against arrhythmias in Kv4.2W362F dominant negative mice, despite QT prolongation. Circulation. 1999; 100: I-769.Abstract.