Accessory Kv1 Subunits Differentially Modulate the Functional Expression of Voltage-Gated K+ Channels in Mouse Ventricular Myocytes
http://www.100md.com
循环研究杂志 2005年第3期
the Department of Molecular Biology and Pharmacology (F.A., J.M.N.), Washington University School of Medicine, St Louis, Mo
Wyeth-Ayerst Research (S.P.K., K.J.R.), Princeton, NJ.
Abstract
Voltage-gated K+ (Kv) channel accessory () subunits associate with pore-forming Kv subunits and modify the properties and/or cell surface expression of Kv channels in heterologous expression systems. There is very little presently known, however, about the functional role(s) of Kv subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kv1 gene (Kv1eC/eC), the studies here were undertaken to explore directly the role of Kv1 in the generation of ventricular Kv currents. Action potential waveforms and peak Kv current densities are indistinguishable in myocytes isolated from the left ventricular apex (LVA) of Kv1eC/eC and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean±SEM Ito,f density is significantly (P0.01) lower in Kv1eC/eC (21.0±0.9 pA/pF; n=68), than in WT (25.3±1.4 pA/pF; n=42), LVA myocytes, and that mean±SEM IK,slow density is significantly (P0.01) higher in Kv1eC/eC (19.1±0.9 pA/pF; n=68), compared with WT (15.9±0.7 pA/pF; n=42), LVA cells. Pharmacological studies demonstrated that the TEA-sensitive component of IK,slow, IK,slow2, is selectively increased in Kv1eC/eC LVA myocytes. In parallel with the alterations in Ito,f and IK,slow2 densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kv1eC/eC ventricles. Taken together, these results demonstrate that Kv1 differentially regulates the functional cell surface expression of myocardial Ito,f and IK,slow2 channels.
Key Words: potassium channels Kv accessory subunits Kv Ito,f IK,slow
Introduction
Voltage-gated K+ (Kv) channels control the amplitudes and durations of myocardial action potentials, and in most cells, multiple Kv channel types are expressed.1 Over the last decade, considerable progress has been made in characterizing the properties of myocardial Kv channels and in defining the roles of individual Kv channel pore-forming () subunits in the generation of these channels.1 In mouse ventricular myocytes, for example, four kinetically distinct Kv currents, Ito,f, Ito,s, IK,slow, and Iss have been identified.2 The fast transient outward K+ current, Ito,f, is encoded by members of the Kv4 subfamily,3 and Kv1.4 underlies Ito,s.4 In addition, IK,slow has been shown to reflect the expression of two molecularly distinct components, IK,slow1 and IK,slow2.5eC8 The 4-aminopyridineeCsensitive component of IK,slow, IK,slow1, is encoded by Kv1.5,5,6 whereas the tetraethylammonium-sensitive component, IK,slow2, is encoded by Kv2.1.7,8 The molecular identity of Iss remains to be determined.
A number of Kv channel accessory subunits, including MinK/MIRPs,9 Kv s,10 KChAP,11 KChIPs,12 and DPPX,13 have also been identified and suggested to play roles in the generation of functional Kv channels. First identified in brain in association with Kv1 subunits,14 the Kv subunits are cytoplasmic proteins with sequence homology to aldo-keto reductases.15 There are three Kv subunit genes, KCNAB1, KCNAB2, and KCNAB3, and each is alternatively spliced to generate (Kv1.x, Kv2.x, and Kv3.x) proteins with unique N termini.10 The C-terminal "core" domains, which mediate interaction with the tetramerization (T1) domain of Kv1 subunits,16 are similar. The interaction between Kv1 and Kv subunits occurs in the endoplasmic reticulum early in channel biosynthesis17 and results in increased trafficking of assembled Kv1-encoded channels to the plasma membrane. Association with Kv subunits also modulates the properties of Kv1 channels.10 Members of the Kv1 subfamily (and Kv3.1) have long N termini that function, similar to the Shaker K+ channel inactivation gate,18 to mediate fast inactivation. Although originally thought to be Kv1 subfamilyeCspecific,19 in heterologous systems, Kv subunits also interact with Kv subunits in other subfamilies,20,21 as well as with KChAP.22
The experiments in this study were undertaken to explore directly the role of Kv1 in the generation of functional myocardial Kv channels and to test the specific hypothesis that Kv1 plays a role in the generation of mouse ventricular Ito,s (Kv1.4)4 or IK,slow1 (Kv1.5)5,6 channels. Electrophysiological experiments on ventricular myocytes isolated from mice bearing a targeted disruption of the Kv1 gene (Kv1eC/eC), however, reveal that Ito,f densities are decreased and IK,slow2 densities are increased compared with the current densities in wild-type cells. Neither of the Kv1 subuniteCencoded currents are altered in Kv1eC/eC myocytes. In addition, biochemical studies reveal that the deletion of Kv1 results in decreased membrane expression of Kv4.3 and increased membrane expression of Kv2.1. These results demonstrate that Kv1 differentially regulates the functional cell surface expression of mouse ventricular Ito,f and IK,slow2 channels.
Materials and Methods
Animals used in the studies here were handled in accordance with The Guide for the Care and Use of Laboratory Animals (National Institutes of Health), and all protocols were approved by the Washington University Animal Studies Committee. The generation of the Kv1eC/eC mice (S.P. Kwak and K.J. Rhodes, unpublished data) and all of the methods used in the present study have been described previously,2,6,24,25 and further details can be found in the expanded Materials and Methods in the online data supplement available at http://circres.ahajournals.org.
Results
Kv Subunit Expression in Adult Mouse Ventricles
The intron-exon structure of the mouse Kv1 (mKCNAB1) gene is illustrated in Figure 1A. The open reading frame comprises 14 exons, and alternative splicing occurs in exon 1 to produce three distinct Kv1 messages, mKv1.1, mKv1.2, and mKv1.3, which on translation, yield Kv1 subunit proteins with unique N termini (Figure 1B). To examine the expression profiles of the Kv1 subunits in adult mouse ventricles (and brains), PCR primers were designed against the common Kv1 core and against the unique N termini of Kv1.1, Kv1.2, and Kv1.3. RT-PCR analyses revealed that the Kv1 core, as well as the Kv1.1 and Kv1.2 splice variants, are readily detected in wild-type (WT) ventricles (Figure 2A). Kv2.1 and Kv3.1 are also expressed in WT ventricles, whereas no Kv1.3 is detected. In adult mouse brain, all three Kv1 splice variants, Kv2.1 and Kv3.1 are expressed (Figure 2A).
RT-PCR analysis of extracts from Kv1eC/eC ventricles and brains revealed that none of the Kv1 splice variants nor the Kv1 core is detected (Figure 2A), consistent with the loss of Kv1. In contrast, both Kv2.1 and Kv3.1 are present in Kv1eC/eC ventricles/brains (Figure 2A). In WT mouse ventricles, the Kv1.1 and Kv1.2 proteins, as well as Kv2, are readily detected (Figure 2B). In contrast, neither Kv1.1 nor Kv1.2 is detected in Western blots of ventricular or brain lysates from Kv1eC/eC animals (Figure 2C).
Functional Consequences of the Targeted Disruption of Kv1
To examine the functional consequences of the targeted disruption of KCNAB1, telemetric ECG recordings were obtained from WT and Kv1eC/eC animals. As evident in the representative recordings in Figure 3A, the morphologies of the QRS complexes and P waves are similar in WT and Kv1eC/eC animals. There were no significant differences in the durations of the QT, PR, QRS, or RR intervals in WT and Kv1eC/eC mice (online Table OS1, available in the online data supplement). Mean±SEM heart rates of WT and Kv1eC/eC animals are indistinguishable, and corrected QT (QTc) intervals in Kv1eC/eC (52±1 ms; n=12) and WT (47±1 ms; n=11) animals, therefore, are not significantly different (online Table OS1). Analysis of the ECG records obtained from Kv1eC/eC animals during 48 hours of continuous monitoring also revealed no evidence of rhythm disturbances (not illustrated).
The similarities in QT (QTc) intervals in Kv1eC/eC and WT animals suggested that repolarization is not affected significantly by the targeted disruption of Kv1. Consistent with this hypothesis, action potential waveforms in LVA myocytes isolated from Kv1eC/eC and WT hearts are not significantly different (Figure 3B): mean±SEM APD90 in WT and Kv1eC/eC LVA myocytes were 17.1±1.5 ms (n=8) and 15.5±4.0 ms (n=9), respectively (online Table OS1).
Voltage-Gated K+ Currents Are Altered in Kv1eC/eC Ventricular Myocytes
As illustrated in Figure 4A, whole-cell voltage-gated K+ (Kv) currents recorded from WT and Kv1eC/eC LVA myocytes are similar. No differences in peak Kv current (Ipeak) densities are observed (Figure 4B). At +40 mV, for example, mean±SEM Ipeak densities were 50.9±1.9 (n=42) and 47.1±1.7 pA/pF (n=68) in WT and Kv1eC/eC LVA myocytes, respectively (Table). Nevertheless, visual inspection of the records suggested that Kv current waveforms in Kv1eC/eC and WT LVA myocytes are distinct. Kinetic analysis of the decay phases of the currents indeed revealed that Ito,f densities in Kv1eC/eC LVA myocytes are significantly (P<0.01) lower than in WT LVA myocytes (Figure 4C). At +40 mV, for example, mean±SEM Ito,f densities were 25.3±1.4 and 21.0±0.9 pA/pF in WT and Kv1eC/eC LVA myocytes, respectively. The voltage dependence of Ito,f activation and the kinetic properties of Ito,f in Kv1eC/eC and WT LVA myocytes are similar (Table). Steady-state inactivation of Ito,f, however, is shifted by eC10 mV in Kv1eC/eC LVA myocytes (Table).
Analysis of the Kv currents (Figure 4A) also revealed that mean±SEM IK,slow density is significantly (P<0.01) higher in Kv1eC/eC, than in WT LVA cells (Figure 4D). At +40 mV, for example, mean±SEM IK,slow densities in Kv1eC/eC and WT LVA cells were 19.1±0.9 and 15.9±0.7 pA/pF, respectively (Table). Iss densities are similar in Kv1eC/eC and WT LVA cells (Figure 4E and Table). Although IK,slow densities are increased, the kinetics of IK,slow inactivation and recovery from inactivation are similar in Kv1eC/eC and WT LVA myocytes (Table). The voltage-dependence of IK,slow inactivation, however, is also shifted (P0.05) by eC10 mV in Kv1eC/eC, compared with WT (n=12) LVA myocytes (Table).
Targeted Disruption of Kv1 Reduces Ito,f and Augments IK,slow2
It has been demonstrated previously that Kv current components can be separated pharmacologically.2,4eC6,8 Mouse ventricular Ito,f, for example, is selectively reduced by nmol/L concentrations of Heteropodatoxin-2/3 (HpTx-2/3), whereas IK,slow is attenuated preferentially by 4-AP and TEA.5eC8 Subsequent experiments, therefore, were focused on quantifying the effects of the targeted disruption of Kv1 on Ito,f and IK,slow. Consistent with the kinetic analysis, the density of the 100 nmol/L HpTx-3eCsensitive current is decreased significantly (P<0.01) in Kv1eC/eC LVA myocytes (Figure 5A).
Previous studies have demonstrated that mouse ventricular IK,slow reflects the expression of two molecularly distinct components. The eol/L 4-APeCsensitive component of IK,slow, IK,slow1, is encoded by Kv1.5,5,6 and the mmol/L TEA-sensitive component, IK,slow2, is encoded by Kv2.1.7,8 To determine whether IK,slow1 and/or IK,slow2 is affected by the targeted disruption of Kv1, the effects of 50 eol/L 4-AP-and 5 mmol/L TEA were determined and compared. No significant differences in the densities of the 50 eol/L 4-APeCsensitive IK,slow1 were observed, whereas the density of the 5 mmol/L TEA-sensitive current, IK,slow2, is significantly (P<0.01) higher in Kv1eC/eC (n=12), than in WT (n=8) LVA myocytes (Figure 5B). Neither the voltage-dependences nor the kinetics of IK,slow1 or IK,slow2 activation or inactivation are measurably affected by the elimination of Kv1.
Similar experiments were completed on isolated myocytes from the septum of WT (n=12) and Kv1eC/eC (n=8) hearts and identical results were obtained: Ito,f densities are reduced and IK,slow2 densities are increased in Kv1eC/eC, compared with WT, septum cells. Similar to the findings for the Kv1.5-encoded IK,slow1 channels (Figure 5B), the densities and properties of the Kv1.4-encoded Ito,s channels are unaffected by the targeted disruption of Kv1.
Kv1/Kv Subunit Interactions in Adult Mouse Ventricles
In heterologous expression systems, it has been demonstrated that Kv and subunits associate.17,19 To determine whether Kv1 subunits interact directly with Kv subunits in adult mouse ventricles, immunoprecipitations using a Kv1.1-specific monoclonal antibody were performed. As illustrated in Figure 6, the anti-Kv1.1 antibody reliably immunoprecipitates Kv1.1. Western blot analysis also revealed that Kv1.2, Kv4.2, and Kv4.3 coimmunoprecipitate with Kv1.1 (Figure 6). In addition, there is very little Kv4.2 or Kv4.3 remaining in the supernatants, suggesting that most of the Kv4.2 and Kv4.3 coimmunoprecipitate with Kv1.1 (Figure 6). In previous studies, we demonstrated that another Kv channel accessory subunit, KChIP2,12 coimmunoprecipitates with Kv4.2 and Kv4.3,24 consistent with a role for KChIP2 in the generation of cardiac Ito,f channels. As would be expected, KChIP2 also coimmunoprecipitates with the anti-Kv1.1 antibody (online Figure OS1, available in the online data supplement).
In contrast to the findings with Kv4.2, Kv4.3, and KChIP2, neither Kv1.5 nor Kv2.1 coimmunoprecipitates with Kv1.1; both Kv1.5 and Kv2.1 are found only in the supernatants (Figure 6). Similar results were obtained using an antieCKv1.2-specific antibody. Parallel immunoprecipitation experiments using anti-Kv1.5 and anti-Kv2.1 antibodies revealed that, although both reliably immunoprecipitate the targeted (Kv1.5 or Kv2.1) proteins, neither Kv1.1 nor Kv1.2 was detected in the immunoprecipitated samples (not illustrated). In addition, immunoprecipitations with the anti-Kv1.1 antibody, followed by Western blot analysis with antibodies targeted against two additional Kv subunits of the Kv1 subfamily, Kv1.2 and Kv1.4, that are also expressed in adult mouse ventricles,4 revealed that neither Kv1.4 nor Kv1.2 appears to associate with Kv1.1 (online Figure OS2). Both Kv1.2, which is of unknown function, and Kv1.4, which encodes Ito,s,4 are found in the supernatants after immunoprecipitations with anti-Kv1.1 (online Figure OS2).
Targeted Disruption of Kv1 Alters the Membrane Expression of Kv Subunits
Similar to other accessory subunits,11,12 Kv subunits have chaperone-like effects, regulating the cell surface expression of Kv subuniteCencoded K+ channels.10,17 Consistent with a chaperone function, Western blot analysis revealed that Kv4.3 membrane expression is decreased significantly (P0.01) and that Kv2.1 membrane expression is increased significantly (P0.001) in Kv1eC/eC ventricles (Figure 7). Given that Kv1 subunits do not appear to interact directly with Kv2.1 (Figure 6), these results suggest that Kv1 exerts an indirect effect on mouse ventricular Kv2.1 expression. There are no significant differences in Kv1.5 membrane expression in WT and Kv1eC/eC ventricles (Figure 7).
Discussion
Targeted Disruption of Kv1 Attenuates Mouse Ventricular Ito,f
The results presented here demonstrate that Ito,f densities are markedly reduced in Kv1eC/eC ventricular myocytes. The biochemical studies demonstrate that Kv4.2 and Kv4.3, as well as Kv1.2, coimmunoprecipitate with Kv1.1, and that the targeted disruption of Kv1 results in decreased membrane expression of Kv4.3. It has been reported previously that the K+ channel interacting protein, KChIP2, binds to Kv4 subunits and modulates the properties of Kv4-encoded K+ currents.12 In addition, KChIP2 coimmunoprecipitates with Kv4.2 and Kv4.3, results interpreted as suggesting that functional mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2, Kv4.3, and KChIP2.25
In heterologous systems, Kv4-encoded currents are modulated by coexpression of a variety of Kv accessory subunits, including Kv1.2,20 Kv3,21 MiRP1,26 KChAP,22 DPPX,13 as well as by NCS1,27 the voltage-gated Na+ (Nav) channel 1 subunit,28 and the scaffolding protein, PSD-95.29 Coexpression with Kv1 or Kv3, for example, increases the cell surface expression of Kv4.3-encoded channels,21 whereas coexpression with Kv1.2 modulates the properties, but not the expression, of Kv4.2-encoded channels.20 In addition, biochemical studies have revealed that Kv1 subunits associate with Kv4 subunits in COS-1 cells,21 as well as in (rat) brain.30 The results presented here suggest that Kv1 subunits contribute to the generation of mouse ventricular Ito,f channels by influencing the membrane expression of Kv4.3. The Kv1 C terminal "core" domain has been shown to interact with Kv1 subunit N terminal T1 domains,16 and recent studies suggest that Kv4 subunit N termini structurally resemble Kv1 T1 domains.31 It seems reasonable to suggest, therefore, that the Kv4.3 N terminus may be important in mediating the interaction with Kv1. The N termini of Kv4 subunits also interact with KChIPs,12,31 suggesting that these domains are multifunctional, mediating subunit interactions and association with KChIP, as well, perhaps, as with Kv1 subunits. It has also been reported, however, that Kv1 subunits regulate the expression of Kv4.3-encoded K+ channels in HEK-293 cells through interactions with the C, not the N, terminus.21 Although the biochemical data presented in this study demonstrate that Kv4.3, as well as Kv4.2 and Kv1.2, coimmunoprecipitate with Kv1.1, it is possible that the interactions between these subunits are indirect, mediated, for example, by other cytoplasmic accessory subunits, such as KChAP,22 the KChIPs, or through scaffolding proteins29 or components of the cytoskeleton.32,33 Interestingly, however, recent studies suggest that Ito,f is also reduced in LVA myocytes isolated from mice in which only the N terminal Kv1.1 inactivation domain is removed, suggesting that the N-terminal domain of Kv1.1 mediates the interaction with Kv4 subunits. Clearly, further studies will be necessary to provide detailed insights into the mechanisms involved in regulating the interactions between Kv1 and Kv4 subunits and the roles of these interactions in controlling the functional cell surface expression of mouse ventricular Ito,f channels.
Targeted Deletion of Kv1 Augments Mouse Ventricular IKslow,2
Because Kv subunits have long been thought to interact specifically with subunits of the Kv1 subfamily,19 the working hypothesis at the outset here was that Kv1 likely associates with Kv1.5 and/or Kv1.4 and participates in the generation of mouse ventricular IK,slow1 and/or Ito,s. No measurable effects on mouse ventricular IK,slow1 or Ito,s, however, were evident in Kv1eC/eC myocytes. In addition, the biochemical data suggest that (mouse) ventricular Kv1 does not associate with either Kv1.5 or Kv1.4. The results presented in this study, therefore, suggest that Kv1 subunits do not function as accessory proteins in the generation of mouse ventricular IK,slow1 or Ito,s channels. The lack of association between Kv1.5 and Kv1.1 in adult mouse ventricles was unexpected given the previous findings that Kv subunits associate with Kv1.5 in human heart.22 Comparison of the amino acid sequences of mouse and human Kv1.5 reveals nearly 90% sequence identity (online Figure OS3). The greatest sequence divergence is in the N termini of these proteins, and future experiments, focused on determining the role(s) of these amino acid differences in the regulation of interactions with Kv1 subunits, are clearly warranted to define the molecular basis of these disparate experimental observations.
Unexpectedly, the experiments here revealed that IK,slow2 densities and the membrane expression of Kv2.1 are increased in Kv1eC/eC ventricles. The biochemical data, however, do not suggest direct interaction(s) between Kv2.1 and Kv1.1 (or Kv1.2), at least in adult mouse ventricles. These results are consistent with previous studies that have failed to identify any interactions between Kv1 and Kv2.1 in (rat) ventricle22 or brain.30 The possibility that Kv1 subunits might play an indirect role in the regulation of Kv2.1 channels, however, is suggested by the observation that Kv1.2 suppresses the modulatory effects of KChAP on Kv2.1-encoded K+ current expression in Xenopus oocytes.22 The upregulation of IK,slow2 and Kv2.1 membrane expression demonstrated here suggest that endogenous Kv1 is a negative modulator of functional mouse ventricular IK,slow2 channel cell surface expression. Taken together, the results also strongly suggest that, in vivo, additional proteins (possibly KChAP22) are involved in the generation of IK,slow2 channels.
Relationship to Previous Studies
Myocardial Kv currents control action potential repolarization and, thus, regulate ventricular diastole. In the diseased myocardium and in experimental (animal) models of cardiac disease, Kv currents are reduced, resulting in increased action potential durations and QT intervals. The attenuation of functional transient3 or delayed rectifier5,7,8 Kv currents in mouse ventricles is also associated with action potential and QT prolongation. The fact that action potential waveforms and QTc intervals in Kv1eC/eC and WT myocytes/animals are indistinguishable likely reflects the fact that Ito,f and IK,slow2 densities are differentially affected by the loss of Kv1, ie, the increase in IK,slow2 appears to compensate for the decrease in Ito,f.
Although functional Kv channels were once thought to reflect the simple tetrameric association of Kv subunits from the same subfamily, a number of Kv accessory subunits and other regulatory proteins are now thought to participate in the generation of native Kv channels. Indeed, several lines of evidence suggest that accessory subunits are required to recapitulate in vitro the properties of native Kv currents.12,13,24 The role of Kv1.1 has been examined previously in studies on mice lacking Kv1.1 only (Kv1.1eC/eC).34 Phenotypic characterization of these mice suggested impaired learning and memory, and electrophysiological studies on Kv1.1eC/eC CA1 pyramidal neurons revealed that action potentials are prolonged and after-hyperpolarizations are reduced.34 Voltage-clamp recordings demonstrated a decrease in the amplitude of the rapidly activating and inactivating Kv current, IA, in isolated Kv1.1eC/eC CA1 cells.34 Although these observations were interpreted as reflecting a change in the rate of inactivation of an A current encoded by Kv1.4,34 this seems unlikely given that considerable evidence now suggests that Kv4 subunits encode neuronal IA channels.35,36 Interestingly, the decrease in IA in Kv1.1eC/eC CA1 neurons is accompanied by an increase in the sustained outward K+ current, ISO,34 which likely is encoded by Kv2.1.37 It would clearly be interesting to determine whether the changes in IA and ISO densities reflect changes in the membrane expression of Kv4.x and/or Kv2.1 subunits in CA1 neurons.
The results presented here demonstrate that Kv1 participates in regulating the functional cell surface expression of native myocardial Ito,f and IK,slow2 channels. Future experiments, using short interfering silencing RNAs (siRNAs) specific for Kv1.1 (or Kv1.2) in WT ventricular myocytes or expression of K1.1 (or Kv1.2) in Kv1eC/eC ventricular myocytes will provide insights into the specific role of these splice variants in the regulation of Ito,f and IK,slow2 channels. In these, as in future studies focused on exploring the detailed molecular mechanisms controlling the functional cell surface expression of cardiac Kv channels and the specific role(s) of Kv1 subunits, the Kv1eC/eC mice will be very useful.
Acknowledgments
Financial support provided by the National Institutes of Health (HL-034161 and HL-066388 to J.M.N.) and the Heartland Affiliate of the American Heart Association (Postdoctoral Fellowship to F.A.) is gratefully acknowledged. We thank Rick Wilson for expert technical assistance in the screening and maintenance of the Kv1eC/eC mice and Drs Hullin Li and Kathryn Yamada for many helpful discussions.
References
Nerbonne JM, Kass RS. Physiology and molecular biology of ion channels contributing to ventricular repolarization. In: Gussak I, ed. Contemporay Cardiology: Cardiac Repolarization: Bridging Basic and Clinical Science. Totowa: Humana Press; 2003: 25eC61.
Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661eC678.
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 alpha subunit. Circ Res. 1998; 83: 560eC567.
Guo W, Xu H, London B, Nerbonne JM. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol. 1999; 521: 587eC599.
London B, Guo W, Pan XH, Lee JS, Shusterman V, Logothetis DA, Nerbonne JM, Hill JA. Targeted replacements of Kv1.5 in the mouse leads to loss of the 4-aminoyridine-sensitive component of IK,slow and resistance to drug induced QT prolongation. Circ Res. 2001; 88: 940eC946.
Li H, Guo W, Yamada KA, Nerbonne JM. Selective elimination of the 4-AP-sensitive component of delayed rectification, IK,slow1, in mouse ventricular myocytes expressing a dominant negative Kv1.5 alpha subunit. Am J Physiol. 2004; 286: H319eCH328.
Zhou J, Kodirov S, Murata M, Buckett PD, Nerbonne JM, Koren G. Regional upregulation of Kv2.1-encoded current, IK,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice. Am J Physiol. 2003; 284: H491eCH500.
Xu H, Barry DM, Li H, Brunet S, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res. 1999; 85: 623eC633.
Abbot GW, Goldstein SA. A superfamily of small potassium channel subunits; form and function of the Mink-related peptides (MiRPs). Quart Rev Biophys. 1998; 31: 357eC398.
Pongs O, Leicher T, Berger M, Roeper J, Bahring R, Wray D, Giese KP, Silva AJ, Storm JF. Functional and molecular aspects of voltage-gated K+ channel beta subunits. Ann NY Acad Sci. 1999; 868: 344eC355.
Wible BA, Yang Q, Kuryshev YAS, Accili EA, Brown AM. Cloning and expression of a novel K+ channel regulatory protein, KChAP. J Biol Chem. 1998; 273: 11745eC11751.
An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature. 2000; 403: 553eC556.
Nadal MS, Ozaita A, Amarillo Y, de Miera EV, Ma Y, Mo W, Goldberg EM, Misumi Y, Ikehara Y, Neubert TA, Rudy B. The CVD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron. 2003; 37: 449eC461.
Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JQ, Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit. Nature. 1994; 369: 289eC294.
Chouinard SW, Wilson GF, Schlimgen AK, Ganetzky B. A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci U S A. 1995; 92: 6763eC6767.
Gulbis JM, Zhou M, Mann S, MacKinnon R. Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels. Science. 2000; 289: 123eC127.
Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS. Beta subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron. 1996; 16: 843eC852.
Hoshi T, Zagotta WN, Aldrich RW. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990; 250: 533eC538.
Sewing S, Roeper J, Pongs O. Kv beta 1 subunit binding specific for Shaker-related potassium channel alpha subunits. Neuron. 1996; 16: 455eC463.
Perez-Garcia MT, Lopez-Lopez JR, Gonzalez C. Kv beta 1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to Kv4.2 but not to Shaker channels. J Gen Physiol. 1999; 113: 897eC907.
Yang EK, Alvira MR, Levitan ES, Takimoto K. Kv beta subunits increase expression of Kv4.3 channels by interacting with their C termini. J Biol Chem. 2001; 276: 4839eC4844.
Kuryshev YA, Wible BA, Gudz TI, Ramirez AN, Brown AM. KChAP/Kv beta 1.2 interactions and their effects on cardiac Kv channel expression. Am J Physiol. 2001; 281: C290eCC299.
Deleted in proof.
Guo W, Li H, Aimond F, Johns DC, Rhodes KJ, Trimmer JS, Nerbonne JM. Role of heteromultimers in the generation of myocardial transient outward K+ currents. Circ Res. 2002; 90: 586eC593.
Brunet SD, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J Physiol. 2004; 559: 103eC120.
Zhang M, Jiang M, Tseng GN. MinK related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as a beta subunit of cardiac transient outward current channel Circ Res. 2001; 88: 1012eC1019.
Guo W, Malin SA, Johns DC, Jeromin A, Nerbonne JM. Modulation of Kv4-encoded K+ currents in the mammalian myocardium by neuronal calcium sensor-1. J Biol Chem. 2002; 277: 26436eC26443.
Deschenes I, Tomaselli GF. Modulation of Kv4.3 current by accessory subunits. FEBS Lett. 2002; 528: 183eC188.
Wong W, Newel EW, Jugloff DG, Jones OT, Schlichter LC. Cell surface targeting and clustering interactions between heterologously expressed PSD-95 and the Shal voltage-gated potassium channel, Kv4.2. J Biol Chem. 2002; 277: 20423eC20430.
Rhodes KJ, Strassle BW, Monagham MM, Bekele-Arcuri Z, Matos MF, Trimmer JS. Association and colocalization of Kv beta1 and Kv beta2 with Kv1 alpha subunits in mammalian brain K+ channel complexes. J Neurosci. 1997; 17: 8246eC8258.
Scannevin RH, Wang K, Jow F, Megules J, Kopsco DC, Edris W, Carroll KC, Lu Q, Xu W, Xu Z, Katz AH, Olland S, Lin L, Taylor M, Stahl M, Malakian K, Somers W, Mosyak L, Bowlby MR, Chanda P, Rhodes KJ. Two N-terminal domains of Kv4 K+ channels regulate binding to and modulation by KChIP1. Neuron. 2004; 41: 587eC598.
Petrecca K, Miller DM, Shrier A. Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin binding protein filamin. J Neurosci. 2000; 20: 8736eC8740.
Wang Z, Eldstrom JR, Jantzi J, Moore ED, Fedida D. Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization. Am J Physiol. 2004; 286: H749eCH759.
Giese KP, Storm JF, Reuter D, Fedorov NB, Shao LR, Leicher T, Pongs O, Silva AJ. Reduced K+ channel inactivation, spike broadening, and after-hyperpolarization in Kv beta1.1-deficient mice with impaired learning. Learn Mem. 1998; 5: 257eC273.
Song W-J, Thatch T, Barananskas G, Ichinohe N, Kitai ST, Surmeier DJ. Somatodendritic depolarization-activated potassium currents in rat neostriatal cholinergic interneurons are predominately of the A type and attributable to co-expression of Kv4.2 and Kv4.1 subunits. J Neurosci. 1998; 18: 3124eC3137.
Malin S, Nerbonne JM. Elimination of the fast transient outward K+ current in superior cervical ganglion cells: molecular dissection of IA. J Neurosci. 2000; 20: 5191eC5199.
Antonucci DE, Lim ST, Vassanelli S, Trimmer JS. Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons. Neuroscience. 2001; 108: 69eC81.(Franck Aimond, Seung P. K)
Wyeth-Ayerst Research (S.P.K., K.J.R.), Princeton, NJ.
Abstract
Voltage-gated K+ (Kv) channel accessory () subunits associate with pore-forming Kv subunits and modify the properties and/or cell surface expression of Kv channels in heterologous expression systems. There is very little presently known, however, about the functional role(s) of Kv subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kv1 gene (Kv1eC/eC), the studies here were undertaken to explore directly the role of Kv1 in the generation of ventricular Kv currents. Action potential waveforms and peak Kv current densities are indistinguishable in myocytes isolated from the left ventricular apex (LVA) of Kv1eC/eC and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean±SEM Ito,f density is significantly (P0.01) lower in Kv1eC/eC (21.0±0.9 pA/pF; n=68), than in WT (25.3±1.4 pA/pF; n=42), LVA myocytes, and that mean±SEM IK,slow density is significantly (P0.01) higher in Kv1eC/eC (19.1±0.9 pA/pF; n=68), compared with WT (15.9±0.7 pA/pF; n=42), LVA cells. Pharmacological studies demonstrated that the TEA-sensitive component of IK,slow, IK,slow2, is selectively increased in Kv1eC/eC LVA myocytes. In parallel with the alterations in Ito,f and IK,slow2 densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kv1eC/eC ventricles. Taken together, these results demonstrate that Kv1 differentially regulates the functional cell surface expression of myocardial Ito,f and IK,slow2 channels.
Key Words: potassium channels Kv accessory subunits Kv Ito,f IK,slow
Introduction
Voltage-gated K+ (Kv) channels control the amplitudes and durations of myocardial action potentials, and in most cells, multiple Kv channel types are expressed.1 Over the last decade, considerable progress has been made in characterizing the properties of myocardial Kv channels and in defining the roles of individual Kv channel pore-forming () subunits in the generation of these channels.1 In mouse ventricular myocytes, for example, four kinetically distinct Kv currents, Ito,f, Ito,s, IK,slow, and Iss have been identified.2 The fast transient outward K+ current, Ito,f, is encoded by members of the Kv4 subfamily,3 and Kv1.4 underlies Ito,s.4 In addition, IK,slow has been shown to reflect the expression of two molecularly distinct components, IK,slow1 and IK,slow2.5eC8 The 4-aminopyridineeCsensitive component of IK,slow, IK,slow1, is encoded by Kv1.5,5,6 whereas the tetraethylammonium-sensitive component, IK,slow2, is encoded by Kv2.1.7,8 The molecular identity of Iss remains to be determined.
A number of Kv channel accessory subunits, including MinK/MIRPs,9 Kv s,10 KChAP,11 KChIPs,12 and DPPX,13 have also been identified and suggested to play roles in the generation of functional Kv channels. First identified in brain in association with Kv1 subunits,14 the Kv subunits are cytoplasmic proteins with sequence homology to aldo-keto reductases.15 There are three Kv subunit genes, KCNAB1, KCNAB2, and KCNAB3, and each is alternatively spliced to generate (Kv1.x, Kv2.x, and Kv3.x) proteins with unique N termini.10 The C-terminal "core" domains, which mediate interaction with the tetramerization (T1) domain of Kv1 subunits,16 are similar. The interaction between Kv1 and Kv subunits occurs in the endoplasmic reticulum early in channel biosynthesis17 and results in increased trafficking of assembled Kv1-encoded channels to the plasma membrane. Association with Kv subunits also modulates the properties of Kv1 channels.10 Members of the Kv1 subfamily (and Kv3.1) have long N termini that function, similar to the Shaker K+ channel inactivation gate,18 to mediate fast inactivation. Although originally thought to be Kv1 subfamilyeCspecific,19 in heterologous systems, Kv subunits also interact with Kv subunits in other subfamilies,20,21 as well as with KChAP.22
The experiments in this study were undertaken to explore directly the role of Kv1 in the generation of functional myocardial Kv channels and to test the specific hypothesis that Kv1 plays a role in the generation of mouse ventricular Ito,s (Kv1.4)4 or IK,slow1 (Kv1.5)5,6 channels. Electrophysiological experiments on ventricular myocytes isolated from mice bearing a targeted disruption of the Kv1 gene (Kv1eC/eC), however, reveal that Ito,f densities are decreased and IK,slow2 densities are increased compared with the current densities in wild-type cells. Neither of the Kv1 subuniteCencoded currents are altered in Kv1eC/eC myocytes. In addition, biochemical studies reveal that the deletion of Kv1 results in decreased membrane expression of Kv4.3 and increased membrane expression of Kv2.1. These results demonstrate that Kv1 differentially regulates the functional cell surface expression of mouse ventricular Ito,f and IK,slow2 channels.
Materials and Methods
Animals used in the studies here were handled in accordance with The Guide for the Care and Use of Laboratory Animals (National Institutes of Health), and all protocols were approved by the Washington University Animal Studies Committee. The generation of the Kv1eC/eC mice (S.P. Kwak and K.J. Rhodes, unpublished data) and all of the methods used in the present study have been described previously,2,6,24,25 and further details can be found in the expanded Materials and Methods in the online data supplement available at http://circres.ahajournals.org.
Results
Kv Subunit Expression in Adult Mouse Ventricles
The intron-exon structure of the mouse Kv1 (mKCNAB1) gene is illustrated in Figure 1A. The open reading frame comprises 14 exons, and alternative splicing occurs in exon 1 to produce three distinct Kv1 messages, mKv1.1, mKv1.2, and mKv1.3, which on translation, yield Kv1 subunit proteins with unique N termini (Figure 1B). To examine the expression profiles of the Kv1 subunits in adult mouse ventricles (and brains), PCR primers were designed against the common Kv1 core and against the unique N termini of Kv1.1, Kv1.2, and Kv1.3. RT-PCR analyses revealed that the Kv1 core, as well as the Kv1.1 and Kv1.2 splice variants, are readily detected in wild-type (WT) ventricles (Figure 2A). Kv2.1 and Kv3.1 are also expressed in WT ventricles, whereas no Kv1.3 is detected. In adult mouse brain, all three Kv1 splice variants, Kv2.1 and Kv3.1 are expressed (Figure 2A).
RT-PCR analysis of extracts from Kv1eC/eC ventricles and brains revealed that none of the Kv1 splice variants nor the Kv1 core is detected (Figure 2A), consistent with the loss of Kv1. In contrast, both Kv2.1 and Kv3.1 are present in Kv1eC/eC ventricles/brains (Figure 2A). In WT mouse ventricles, the Kv1.1 and Kv1.2 proteins, as well as Kv2, are readily detected (Figure 2B). In contrast, neither Kv1.1 nor Kv1.2 is detected in Western blots of ventricular or brain lysates from Kv1eC/eC animals (Figure 2C).
Functional Consequences of the Targeted Disruption of Kv1
To examine the functional consequences of the targeted disruption of KCNAB1, telemetric ECG recordings were obtained from WT and Kv1eC/eC animals. As evident in the representative recordings in Figure 3A, the morphologies of the QRS complexes and P waves are similar in WT and Kv1eC/eC animals. There were no significant differences in the durations of the QT, PR, QRS, or RR intervals in WT and Kv1eC/eC mice (online Table OS1, available in the online data supplement). Mean±SEM heart rates of WT and Kv1eC/eC animals are indistinguishable, and corrected QT (QTc) intervals in Kv1eC/eC (52±1 ms; n=12) and WT (47±1 ms; n=11) animals, therefore, are not significantly different (online Table OS1). Analysis of the ECG records obtained from Kv1eC/eC animals during 48 hours of continuous monitoring also revealed no evidence of rhythm disturbances (not illustrated).
The similarities in QT (QTc) intervals in Kv1eC/eC and WT animals suggested that repolarization is not affected significantly by the targeted disruption of Kv1. Consistent with this hypothesis, action potential waveforms in LVA myocytes isolated from Kv1eC/eC and WT hearts are not significantly different (Figure 3B): mean±SEM APD90 in WT and Kv1eC/eC LVA myocytes were 17.1±1.5 ms (n=8) and 15.5±4.0 ms (n=9), respectively (online Table OS1).
Voltage-Gated K+ Currents Are Altered in Kv1eC/eC Ventricular Myocytes
As illustrated in Figure 4A, whole-cell voltage-gated K+ (Kv) currents recorded from WT and Kv1eC/eC LVA myocytes are similar. No differences in peak Kv current (Ipeak) densities are observed (Figure 4B). At +40 mV, for example, mean±SEM Ipeak densities were 50.9±1.9 (n=42) and 47.1±1.7 pA/pF (n=68) in WT and Kv1eC/eC LVA myocytes, respectively (Table). Nevertheless, visual inspection of the records suggested that Kv current waveforms in Kv1eC/eC and WT LVA myocytes are distinct. Kinetic analysis of the decay phases of the currents indeed revealed that Ito,f densities in Kv1eC/eC LVA myocytes are significantly (P<0.01) lower than in WT LVA myocytes (Figure 4C). At +40 mV, for example, mean±SEM Ito,f densities were 25.3±1.4 and 21.0±0.9 pA/pF in WT and Kv1eC/eC LVA myocytes, respectively. The voltage dependence of Ito,f activation and the kinetic properties of Ito,f in Kv1eC/eC and WT LVA myocytes are similar (Table). Steady-state inactivation of Ito,f, however, is shifted by eC10 mV in Kv1eC/eC LVA myocytes (Table).
Analysis of the Kv currents (Figure 4A) also revealed that mean±SEM IK,slow density is significantly (P<0.01) higher in Kv1eC/eC, than in WT LVA cells (Figure 4D). At +40 mV, for example, mean±SEM IK,slow densities in Kv1eC/eC and WT LVA cells were 19.1±0.9 and 15.9±0.7 pA/pF, respectively (Table). Iss densities are similar in Kv1eC/eC and WT LVA cells (Figure 4E and Table). Although IK,slow densities are increased, the kinetics of IK,slow inactivation and recovery from inactivation are similar in Kv1eC/eC and WT LVA myocytes (Table). The voltage-dependence of IK,slow inactivation, however, is also shifted (P0.05) by eC10 mV in Kv1eC/eC, compared with WT (n=12) LVA myocytes (Table).
Targeted Disruption of Kv1 Reduces Ito,f and Augments IK,slow2
It has been demonstrated previously that Kv current components can be separated pharmacologically.2,4eC6,8 Mouse ventricular Ito,f, for example, is selectively reduced by nmol/L concentrations of Heteropodatoxin-2/3 (HpTx-2/3), whereas IK,slow is attenuated preferentially by 4-AP and TEA.5eC8 Subsequent experiments, therefore, were focused on quantifying the effects of the targeted disruption of Kv1 on Ito,f and IK,slow. Consistent with the kinetic analysis, the density of the 100 nmol/L HpTx-3eCsensitive current is decreased significantly (P<0.01) in Kv1eC/eC LVA myocytes (Figure 5A).
Previous studies have demonstrated that mouse ventricular IK,slow reflects the expression of two molecularly distinct components. The eol/L 4-APeCsensitive component of IK,slow, IK,slow1, is encoded by Kv1.5,5,6 and the mmol/L TEA-sensitive component, IK,slow2, is encoded by Kv2.1.7,8 To determine whether IK,slow1 and/or IK,slow2 is affected by the targeted disruption of Kv1, the effects of 50 eol/L 4-AP-and 5 mmol/L TEA were determined and compared. No significant differences in the densities of the 50 eol/L 4-APeCsensitive IK,slow1 were observed, whereas the density of the 5 mmol/L TEA-sensitive current, IK,slow2, is significantly (P<0.01) higher in Kv1eC/eC (n=12), than in WT (n=8) LVA myocytes (Figure 5B). Neither the voltage-dependences nor the kinetics of IK,slow1 or IK,slow2 activation or inactivation are measurably affected by the elimination of Kv1.
Similar experiments were completed on isolated myocytes from the septum of WT (n=12) and Kv1eC/eC (n=8) hearts and identical results were obtained: Ito,f densities are reduced and IK,slow2 densities are increased in Kv1eC/eC, compared with WT, septum cells. Similar to the findings for the Kv1.5-encoded IK,slow1 channels (Figure 5B), the densities and properties of the Kv1.4-encoded Ito,s channels are unaffected by the targeted disruption of Kv1.
Kv1/Kv Subunit Interactions in Adult Mouse Ventricles
In heterologous expression systems, it has been demonstrated that Kv and subunits associate.17,19 To determine whether Kv1 subunits interact directly with Kv subunits in adult mouse ventricles, immunoprecipitations using a Kv1.1-specific monoclonal antibody were performed. As illustrated in Figure 6, the anti-Kv1.1 antibody reliably immunoprecipitates Kv1.1. Western blot analysis also revealed that Kv1.2, Kv4.2, and Kv4.3 coimmunoprecipitate with Kv1.1 (Figure 6). In addition, there is very little Kv4.2 or Kv4.3 remaining in the supernatants, suggesting that most of the Kv4.2 and Kv4.3 coimmunoprecipitate with Kv1.1 (Figure 6). In previous studies, we demonstrated that another Kv channel accessory subunit, KChIP2,12 coimmunoprecipitates with Kv4.2 and Kv4.3,24 consistent with a role for KChIP2 in the generation of cardiac Ito,f channels. As would be expected, KChIP2 also coimmunoprecipitates with the anti-Kv1.1 antibody (online Figure OS1, available in the online data supplement).
In contrast to the findings with Kv4.2, Kv4.3, and KChIP2, neither Kv1.5 nor Kv2.1 coimmunoprecipitates with Kv1.1; both Kv1.5 and Kv2.1 are found only in the supernatants (Figure 6). Similar results were obtained using an antieCKv1.2-specific antibody. Parallel immunoprecipitation experiments using anti-Kv1.5 and anti-Kv2.1 antibodies revealed that, although both reliably immunoprecipitate the targeted (Kv1.5 or Kv2.1) proteins, neither Kv1.1 nor Kv1.2 was detected in the immunoprecipitated samples (not illustrated). In addition, immunoprecipitations with the anti-Kv1.1 antibody, followed by Western blot analysis with antibodies targeted against two additional Kv subunits of the Kv1 subfamily, Kv1.2 and Kv1.4, that are also expressed in adult mouse ventricles,4 revealed that neither Kv1.4 nor Kv1.2 appears to associate with Kv1.1 (online Figure OS2). Both Kv1.2, which is of unknown function, and Kv1.4, which encodes Ito,s,4 are found in the supernatants after immunoprecipitations with anti-Kv1.1 (online Figure OS2).
Targeted Disruption of Kv1 Alters the Membrane Expression of Kv Subunits
Similar to other accessory subunits,11,12 Kv subunits have chaperone-like effects, regulating the cell surface expression of Kv subuniteCencoded K+ channels.10,17 Consistent with a chaperone function, Western blot analysis revealed that Kv4.3 membrane expression is decreased significantly (P0.01) and that Kv2.1 membrane expression is increased significantly (P0.001) in Kv1eC/eC ventricles (Figure 7). Given that Kv1 subunits do not appear to interact directly with Kv2.1 (Figure 6), these results suggest that Kv1 exerts an indirect effect on mouse ventricular Kv2.1 expression. There are no significant differences in Kv1.5 membrane expression in WT and Kv1eC/eC ventricles (Figure 7).
Discussion
Targeted Disruption of Kv1 Attenuates Mouse Ventricular Ito,f
The results presented here demonstrate that Ito,f densities are markedly reduced in Kv1eC/eC ventricular myocytes. The biochemical studies demonstrate that Kv4.2 and Kv4.3, as well as Kv1.2, coimmunoprecipitate with Kv1.1, and that the targeted disruption of Kv1 results in decreased membrane expression of Kv4.3. It has been reported previously that the K+ channel interacting protein, KChIP2, binds to Kv4 subunits and modulates the properties of Kv4-encoded K+ currents.12 In addition, KChIP2 coimmunoprecipitates with Kv4.2 and Kv4.3, results interpreted as suggesting that functional mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2, Kv4.3, and KChIP2.25
In heterologous systems, Kv4-encoded currents are modulated by coexpression of a variety of Kv accessory subunits, including Kv1.2,20 Kv3,21 MiRP1,26 KChAP,22 DPPX,13 as well as by NCS1,27 the voltage-gated Na+ (Nav) channel 1 subunit,28 and the scaffolding protein, PSD-95.29 Coexpression with Kv1 or Kv3, for example, increases the cell surface expression of Kv4.3-encoded channels,21 whereas coexpression with Kv1.2 modulates the properties, but not the expression, of Kv4.2-encoded channels.20 In addition, biochemical studies have revealed that Kv1 subunits associate with Kv4 subunits in COS-1 cells,21 as well as in (rat) brain.30 The results presented here suggest that Kv1 subunits contribute to the generation of mouse ventricular Ito,f channels by influencing the membrane expression of Kv4.3. The Kv1 C terminal "core" domain has been shown to interact with Kv1 subunit N terminal T1 domains,16 and recent studies suggest that Kv4 subunit N termini structurally resemble Kv1 T1 domains.31 It seems reasonable to suggest, therefore, that the Kv4.3 N terminus may be important in mediating the interaction with Kv1. The N termini of Kv4 subunits also interact with KChIPs,12,31 suggesting that these domains are multifunctional, mediating subunit interactions and association with KChIP, as well, perhaps, as with Kv1 subunits. It has also been reported, however, that Kv1 subunits regulate the expression of Kv4.3-encoded K+ channels in HEK-293 cells through interactions with the C, not the N, terminus.21 Although the biochemical data presented in this study demonstrate that Kv4.3, as well as Kv4.2 and Kv1.2, coimmunoprecipitate with Kv1.1, it is possible that the interactions between these subunits are indirect, mediated, for example, by other cytoplasmic accessory subunits, such as KChAP,22 the KChIPs, or through scaffolding proteins29 or components of the cytoskeleton.32,33 Interestingly, however, recent studies suggest that Ito,f is also reduced in LVA myocytes isolated from mice in which only the N terminal Kv1.1 inactivation domain is removed, suggesting that the N-terminal domain of Kv1.1 mediates the interaction with Kv4 subunits. Clearly, further studies will be necessary to provide detailed insights into the mechanisms involved in regulating the interactions between Kv1 and Kv4 subunits and the roles of these interactions in controlling the functional cell surface expression of mouse ventricular Ito,f channels.
Targeted Deletion of Kv1 Augments Mouse Ventricular IKslow,2
Because Kv subunits have long been thought to interact specifically with subunits of the Kv1 subfamily,19 the working hypothesis at the outset here was that Kv1 likely associates with Kv1.5 and/or Kv1.4 and participates in the generation of mouse ventricular IK,slow1 and/or Ito,s. No measurable effects on mouse ventricular IK,slow1 or Ito,s, however, were evident in Kv1eC/eC myocytes. In addition, the biochemical data suggest that (mouse) ventricular Kv1 does not associate with either Kv1.5 or Kv1.4. The results presented in this study, therefore, suggest that Kv1 subunits do not function as accessory proteins in the generation of mouse ventricular IK,slow1 or Ito,s channels. The lack of association between Kv1.5 and Kv1.1 in adult mouse ventricles was unexpected given the previous findings that Kv subunits associate with Kv1.5 in human heart.22 Comparison of the amino acid sequences of mouse and human Kv1.5 reveals nearly 90% sequence identity (online Figure OS3). The greatest sequence divergence is in the N termini of these proteins, and future experiments, focused on determining the role(s) of these amino acid differences in the regulation of interactions with Kv1 subunits, are clearly warranted to define the molecular basis of these disparate experimental observations.
Unexpectedly, the experiments here revealed that IK,slow2 densities and the membrane expression of Kv2.1 are increased in Kv1eC/eC ventricles. The biochemical data, however, do not suggest direct interaction(s) between Kv2.1 and Kv1.1 (or Kv1.2), at least in adult mouse ventricles. These results are consistent with previous studies that have failed to identify any interactions between Kv1 and Kv2.1 in (rat) ventricle22 or brain.30 The possibility that Kv1 subunits might play an indirect role in the regulation of Kv2.1 channels, however, is suggested by the observation that Kv1.2 suppresses the modulatory effects of KChAP on Kv2.1-encoded K+ current expression in Xenopus oocytes.22 The upregulation of IK,slow2 and Kv2.1 membrane expression demonstrated here suggest that endogenous Kv1 is a negative modulator of functional mouse ventricular IK,slow2 channel cell surface expression. Taken together, the results also strongly suggest that, in vivo, additional proteins (possibly KChAP22) are involved in the generation of IK,slow2 channels.
Relationship to Previous Studies
Myocardial Kv currents control action potential repolarization and, thus, regulate ventricular diastole. In the diseased myocardium and in experimental (animal) models of cardiac disease, Kv currents are reduced, resulting in increased action potential durations and QT intervals. The attenuation of functional transient3 or delayed rectifier5,7,8 Kv currents in mouse ventricles is also associated with action potential and QT prolongation. The fact that action potential waveforms and QTc intervals in Kv1eC/eC and WT myocytes/animals are indistinguishable likely reflects the fact that Ito,f and IK,slow2 densities are differentially affected by the loss of Kv1, ie, the increase in IK,slow2 appears to compensate for the decrease in Ito,f.
Although functional Kv channels were once thought to reflect the simple tetrameric association of Kv subunits from the same subfamily, a number of Kv accessory subunits and other regulatory proteins are now thought to participate in the generation of native Kv channels. Indeed, several lines of evidence suggest that accessory subunits are required to recapitulate in vitro the properties of native Kv currents.12,13,24 The role of Kv1.1 has been examined previously in studies on mice lacking Kv1.1 only (Kv1.1eC/eC).34 Phenotypic characterization of these mice suggested impaired learning and memory, and electrophysiological studies on Kv1.1eC/eC CA1 pyramidal neurons revealed that action potentials are prolonged and after-hyperpolarizations are reduced.34 Voltage-clamp recordings demonstrated a decrease in the amplitude of the rapidly activating and inactivating Kv current, IA, in isolated Kv1.1eC/eC CA1 cells.34 Although these observations were interpreted as reflecting a change in the rate of inactivation of an A current encoded by Kv1.4,34 this seems unlikely given that considerable evidence now suggests that Kv4 subunits encode neuronal IA channels.35,36 Interestingly, the decrease in IA in Kv1.1eC/eC CA1 neurons is accompanied by an increase in the sustained outward K+ current, ISO,34 which likely is encoded by Kv2.1.37 It would clearly be interesting to determine whether the changes in IA and ISO densities reflect changes in the membrane expression of Kv4.x and/or Kv2.1 subunits in CA1 neurons.
The results presented here demonstrate that Kv1 participates in regulating the functional cell surface expression of native myocardial Ito,f and IK,slow2 channels. Future experiments, using short interfering silencing RNAs (siRNAs) specific for Kv1.1 (or Kv1.2) in WT ventricular myocytes or expression of K1.1 (or Kv1.2) in Kv1eC/eC ventricular myocytes will provide insights into the specific role of these splice variants in the regulation of Ito,f and IK,slow2 channels. In these, as in future studies focused on exploring the detailed molecular mechanisms controlling the functional cell surface expression of cardiac Kv channels and the specific role(s) of Kv1 subunits, the Kv1eC/eC mice will be very useful.
Acknowledgments
Financial support provided by the National Institutes of Health (HL-034161 and HL-066388 to J.M.N.) and the Heartland Affiliate of the American Heart Association (Postdoctoral Fellowship to F.A.) is gratefully acknowledged. We thank Rick Wilson for expert technical assistance in the screening and maintenance of the Kv1eC/eC mice and Drs Hullin Li and Kathryn Yamada for many helpful discussions.
References
Nerbonne JM, Kass RS. Physiology and molecular biology of ion channels contributing to ventricular repolarization. In: Gussak I, ed. Contemporay Cardiology: Cardiac Repolarization: Bridging Basic and Clinical Science. Totowa: Humana Press; 2003: 25eC61.
Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K+ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661eC678.
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 alpha subunit. Circ Res. 1998; 83: 560eC567.
Guo W, Xu H, London B, Nerbonne JM. Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes. J Physiol. 1999; 521: 587eC599.
London B, Guo W, Pan XH, Lee JS, Shusterman V, Logothetis DA, Nerbonne JM, Hill JA. Targeted replacements of Kv1.5 in the mouse leads to loss of the 4-aminoyridine-sensitive component of IK,slow and resistance to drug induced QT prolongation. Circ Res. 2001; 88: 940eC946.
Li H, Guo W, Yamada KA, Nerbonne JM. Selective elimination of the 4-AP-sensitive component of delayed rectification, IK,slow1, in mouse ventricular myocytes expressing a dominant negative Kv1.5 alpha subunit. Am J Physiol. 2004; 286: H319eCH328.
Zhou J, Kodirov S, Murata M, Buckett PD, Nerbonne JM, Koren G. Regional upregulation of Kv2.1-encoded current, IK,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice. Am J Physiol. 2003; 284: H491eCH500.
Xu H, Barry DM, Li H, Brunet S, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res. 1999; 85: 623eC633.
Abbot GW, Goldstein SA. A superfamily of small potassium channel subunits; form and function of the Mink-related peptides (MiRPs). Quart Rev Biophys. 1998; 31: 357eC398.
Pongs O, Leicher T, Berger M, Roeper J, Bahring R, Wray D, Giese KP, Silva AJ, Storm JF. Functional and molecular aspects of voltage-gated K+ channel beta subunits. Ann NY Acad Sci. 1999; 868: 344eC355.
Wible BA, Yang Q, Kuryshev YAS, Accili EA, Brown AM. Cloning and expression of a novel K+ channel regulatory protein, KChAP. J Biol Chem. 1998; 273: 11745eC11751.
An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature. 2000; 403: 553eC556.
Nadal MS, Ozaita A, Amarillo Y, de Miera EV, Ma Y, Mo W, Goldberg EM, Misumi Y, Ikehara Y, Neubert TA, Rudy B. The CVD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron. 2003; 37: 449eC461.
Rettig J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JQ, Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit. Nature. 1994; 369: 289eC294.
Chouinard SW, Wilson GF, Schlimgen AK, Ganetzky B. A potassium channel beta subunit related to the aldo-keto reductase superfamily is encoded by the Drosophila hyperkinetic locus. Proc Natl Acad Sci U S A. 1995; 92: 6763eC6767.
Gulbis JM, Zhou M, Mann S, MacKinnon R. Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels. Science. 2000; 289: 123eC127.
Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, Trimmer JS. Beta subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron. 1996; 16: 843eC852.
Hoshi T, Zagotta WN, Aldrich RW. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990; 250: 533eC538.
Sewing S, Roeper J, Pongs O. Kv beta 1 subunit binding specific for Shaker-related potassium channel alpha subunits. Neuron. 1996; 16: 455eC463.
Perez-Garcia MT, Lopez-Lopez JR, Gonzalez C. Kv beta 1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to Kv4.2 but not to Shaker channels. J Gen Physiol. 1999; 113: 897eC907.
Yang EK, Alvira MR, Levitan ES, Takimoto K. Kv beta subunits increase expression of Kv4.3 channels by interacting with their C termini. J Biol Chem. 2001; 276: 4839eC4844.
Kuryshev YA, Wible BA, Gudz TI, Ramirez AN, Brown AM. KChAP/Kv beta 1.2 interactions and their effects on cardiac Kv channel expression. Am J Physiol. 2001; 281: C290eCC299.
Deleted in proof.
Guo W, Li H, Aimond F, Johns DC, Rhodes KJ, Trimmer JS, Nerbonne JM. Role of heteromultimers in the generation of myocardial transient outward K+ currents. Circ Res. 2002; 90: 586eC593.
Brunet SD, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J Physiol. 2004; 559: 103eC120.
Zhang M, Jiang M, Tseng GN. MinK related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as a beta subunit of cardiac transient outward current channel Circ Res. 2001; 88: 1012eC1019.
Guo W, Malin SA, Johns DC, Jeromin A, Nerbonne JM. Modulation of Kv4-encoded K+ currents in the mammalian myocardium by neuronal calcium sensor-1. J Biol Chem. 2002; 277: 26436eC26443.
Deschenes I, Tomaselli GF. Modulation of Kv4.3 current by accessory subunits. FEBS Lett. 2002; 528: 183eC188.
Wong W, Newel EW, Jugloff DG, Jones OT, Schlichter LC. Cell surface targeting and clustering interactions between heterologously expressed PSD-95 and the Shal voltage-gated potassium channel, Kv4.2. J Biol Chem. 2002; 277: 20423eC20430.
Rhodes KJ, Strassle BW, Monagham MM, Bekele-Arcuri Z, Matos MF, Trimmer JS. Association and colocalization of Kv beta1 and Kv beta2 with Kv1 alpha subunits in mammalian brain K+ channel complexes. J Neurosci. 1997; 17: 8246eC8258.
Scannevin RH, Wang K, Jow F, Megules J, Kopsco DC, Edris W, Carroll KC, Lu Q, Xu W, Xu Z, Katz AH, Olland S, Lin L, Taylor M, Stahl M, Malakian K, Somers W, Mosyak L, Bowlby MR, Chanda P, Rhodes KJ. Two N-terminal domains of Kv4 K+ channels regulate binding to and modulation by KChIP1. Neuron. 2004; 41: 587eC598.
Petrecca K, Miller DM, Shrier A. Localization and enhanced current density of the Kv4.2 potassium channel by interaction with the actin binding protein filamin. J Neurosci. 2000; 20: 8736eC8740.
Wang Z, Eldstrom JR, Jantzi J, Moore ED, Fedida D. Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization. Am J Physiol. 2004; 286: H749eCH759.
Giese KP, Storm JF, Reuter D, Fedorov NB, Shao LR, Leicher T, Pongs O, Silva AJ. Reduced K+ channel inactivation, spike broadening, and after-hyperpolarization in Kv beta1.1-deficient mice with impaired learning. Learn Mem. 1998; 5: 257eC273.
Song W-J, Thatch T, Barananskas G, Ichinohe N, Kitai ST, Surmeier DJ. Somatodendritic depolarization-activated potassium currents in rat neostriatal cholinergic interneurons are predominately of the A type and attributable to co-expression of Kv4.2 and Kv4.1 subunits. J Neurosci. 1998; 18: 3124eC3137.
Malin S, Nerbonne JM. Elimination of the fast transient outward K+ current in superior cervical ganglion cells: molecular dissection of IA. J Neurosci. 2000; 20: 5191eC5199.
Antonucci DE, Lim ST, Vassanelli S, Trimmer JS. Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons. Neuroscience. 2001; 108: 69eC81.(Franck Aimond, Seung P. K)