This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 96/4/451    most recent 01.RES.0000156890.25876.63v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aimond, F. Articles by Nerbonne, J. M. Search for Related Content PubMed PubMed Citation Articles by Aimond, F. Articles by Nerbonne, J. M. Related Collections Other myocardial biology Animal models of human disease Arrythmias-basic studies

(Circulation Research. 2005;96:451.)
© 2005 American Heart Association, Inc.

Cellular Biology

Accessory Kvß₁ Subunits Differentially Modulate the Functional Expression of Voltage-Gated K⁺ Channels in Mouse Ventricular Myocytes

Franck Aimond, Seung P. Kwak, Kenneth J. Rhodes, Jeanne M. Nerbonne

From the Department of Molecular Biology and Pharmacology (F.A., J.M.N.), Washington University School of Medicine, St Louis, Mo; and Wyeth-Ayerst Research (S.P.K., K.J.R.), Princeton, NJ.

Correspondence to Jeanne M. Nerbonne, Department of Molecular Biology and Pharmacology, Washington University Medical School, 660 South Euclid Ave, Box 8103, St Louis, MO 63110-1093. E-mail jnerbonne{at}msnotes.wustl.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Voltage-gated K⁺ (Kv) channel accessory (ß) 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 ß subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kvß₁ gene (Kvß₁^–/–), the studies here were undertaken to explore directly the role of Kvß₁ 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 Kvß₁^–/– and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean±SEM I_to,f density is significantly (P0.01) lower in Kvß₁^–/– (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 I_K,slow density is significantly (P0.01) higher in Kvß₁^–/– (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 I_K,slow, I_K,slow2, is selectively increased in Kvß₁^–/– LVA myocytes. In parallel with the alterations in I_to,f and I_K,slow2 densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kvß₁^–/– ventricles. Taken together, these results demonstrate that Kvß₁ differentially regulates the functional cell surface expression of myocardial I_to,f and I_K,slow2 channels.

Key Words: potassium channels • Kv accessory subunits • Kvß • I_to,f • I_K,slow

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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, I_to,f, I_to,s, I_K,slow, and I_ss have been identified.² The fast transient outward K⁺ current, I_to,_f, is encoded by members of the Kv4 subfamily,³ and Kv1.4 underlies I_to,s.⁴ In addition, I_K,slow has been shown to reflect the expression of two molecularly distinct components, I_K,slow1 and I_K,slow2.^5–8 The 4-aminopyridine–sensitive component of I_K,slow, I_K,slow1, is encoded by Kv1.5,^5,6 whereas the tetraethylammonium-sensitive component, I_K,slow2, is encoded by Kv2.1.^7,8 The molecular identity of I_ss remains to be determined.

A number of Kv channel accessory subunits, including MinK/MIRPs,⁹ Kv ßs,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 ß subunits are cytoplasmic proteins with sequence homology to aldo-keto reductases.¹⁵ There are three Kv ß subunit genes, KCNAB1, KCNAB2, and KCNAB3, and each is alternatively spliced to generate (Kvß_1.x, Kvß_2.x, and Kvß_3.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 ß 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 ß subunits also modulates the properties of Kv1 channels.¹⁰ Members of the Kvß₁ subfamily (and Kvß_3.1) 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 subfamily–specific,¹⁹ in heterologous systems, Kv ß 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 Kvß₁ in the generation of functional myocardial Kv channels and to test the specific hypothesis that Kvß₁ plays a role in the generation of mouse ventricular I_to,s (Kv1.4)⁴ or I_K,slow1 (Kv1.5)^5,6 channels. Electrophysiological experiments on ventricular myocytes isolated from mice bearing a targeted disruption of the Kvß₁ gene (Kvß₁^–/–), however, reveal that I_to,f densities are decreased and I_K,slow2 densities are increased compared with the current densities in wild-type cells. Neither of the Kv1 subunit–encoded currents are altered in Kvß₁^–/– myocytes. In addition, biochemical studies reveal that the deletion of Kvß₁ results in decreased membrane expression of Kv4.3 and increased membrane expression of Kv2.1. These results demonstrate that Kvß₁ differentially regulates the functional cell surface expression of mouse ventricular I_to,f and I_K,slow2 channels.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 Kvß₁^–/– 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kv ß Subunit Expression in Adult Mouse Ventricles
The intron-exon structure of the mouse Kvß₁ (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 Kvß₁ messages, mKvß_1.1, mKvß_1.2, and mKvß_1.3, which on translation, yield Kvß₁ subunit proteins with unique N termini (Figure 1B). To examine the expression profiles of the Kvß₁ subunits in adult mouse ventricles (and brains), PCR primers were designed against the common Kvß₁ core and against the unique N termini of Kvß_1.1, Kvß_1.2, and Kvß_1.3. RT-PCR analyses revealed that the Kvß₁ core, as well as the Kvß_1.1 and Kvß_1.2 splice variants, are readily detected in wild-type (WT) ventricles (Figure 2A). Kvß_2.1 and Kvß_3.1 are also expressed in WT ventricles, whereas no Kvß_1.3 is detected. In adult mouse brain, all three Kvß₁ splice variants, Kvß_2.1 and Kvß_3.1 are expressed (Figure 2A).

View larger version (51K): [in this window] [in a new window]   Figure 1. KCNAB1 encodes the Kvß1.1, ß1.2, and ß1.3 splice variants. A, Physical map of mouse KCNAB1 is displayed. Exons 2 to 14 are drawn to scale and numbered accordingly. Distances between exon 2 and the splice sites in exon 1 are indicated. In the bottom panel, the map is redrawn to better illustrate the splicing. B, Deduced nucleotide and amino acid sequences of the three mKvß1 splice variants. Alternative amino termini and the beginning of the common Kvß1 core region are illustrated; the complete C-terminal core sequence is available on Genbank (accession number U65591). Dashed lines (-) indicate sequence identity, and the numbers in boxes at the end of each sequence are the lengths of the amino termini in Kvß1.1, Kvß1.2, and Kvß1.3.

View larger version (71K): [in this window] [in a new window]   Figure 2. Kv ß subunit mRNA and protein expression in WT and Kvß1–/– mice. A, Using RT-PCR, Kvß1.1, Kvß1.2, Kvß2.1, and Kvß3.1 are readily detected in WT C57BL6 mouse ventricles and brains; Kvß1.3 is also expressed in brain. Screening with primers designed against the Kvß1 core and the Kvß1.1, Kvß1.2, or Kvß1.3 N-termini revealed that Kvß1 is undetectable in the brains and ventricles of Kvß1–/– animals. Although Kvß1 is eliminated, Kvß2.1 and Kvß3.1 expression appear unaffected. B, Western blots demonstrate Kvß1.1, Kvß1.2, and Kvß2 expression in WT mouse ventricles. Both Kvß1.1 and Kvß1.2 are eliminated in Kvß1–/– ventricles, whereas Kvß2 protein expression is not affected.

RT-PCR analysis of extracts from Kvß₁^–/– ventricles and brains revealed that none of the Kvß₁ splice variants nor the Kvß₁ core is detected (Figure 2A), consistent with the loss of Kvß₁. In contrast, both Kvß_2.1 and Kvß_3.1 are present in Kvß₁^–/– ventricles/brains (Figure 2A). In WT mouse ventricles, the Kvß_1.1 and Kvß_1.2 proteins, as well as Kvß₂, are readily detected (Figure 2B). In contrast, neither Kvß_1.1 nor Kvß_1.2 is detected in Western blots of ventricular or brain lysates from Kvß₁^–/– animals (Figure 2C).

Functional Consequences of the Targeted Disruption of Kvß₁
To examine the functional consequences of the targeted disruption of KCNAB1, telemetric ECG recordings were obtained from WT and Kvß₁^–/– animals. As evident in the representative recordings in Figure 3A, the morphologies of the QRS complexes and P waves are similar in WT and Kvß₁^–/– animals. There were no significant differences in the durations of the QT, PR, QRS, or RR intervals in WT and Kvß₁^–/– mice (online Table OS1, available in the online data supplement). Mean±SEM heart rates of WT and Kvß₁^–/– animals are indistinguishable, and corrected QT (QTc) intervals in Kvß₁^–/– (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 Kvß₁^–/– animals during 48 hours of continuous monitoring also revealed no evidence of rhythm disturbances (not illustrated).

View larger version (21K): [in this window] [in a new window]   Figure 3. ECG and action potential waveforms in WT and Kvß1–/– mice are indistinguishable. A, Representative telemetric ECG recordings from adult WT and Kvß1–/– animals; QT intervals are indicated. No significant differences in heart rates and/or in the morphologies of the P waves, QRS complexes, or QT intervals are evident in recordings from WT and Kvß1–/– animals. B, Action potential waveforms in WT and Kvß1–/– LVA myocytes are similar. Also see online Table OS1.

The similarities in QT (QTc) intervals in Kvß₁^–/– and WT animals suggested that repolarization is not affected significantly by the targeted disruption of Kvß₁. Consistent with this hypothesis, action potential waveforms in LVA myocytes isolated from Kvß₁^–/– and WT hearts are not significantly different (Figure 3B): mean±SEM APD₉₀ in WT and Kvß₁^–/– 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 Kvß₁^–/– Ventricular Myocytes
As illustrated in Figure 4A, whole-cell voltage-gated K⁺ (Kv) currents recorded from WT and Kvß₁^–/– LVA myocytes are similar. No differences in peak Kv current (I_peak) densities are observed (Figure 4B). At +40 mV, for example, mean±SEM I_peak densities were 50.9±1.9 (n=42) and 47.1±1.7 pA/pF (n=68) in WT and Kvß₁^–/– LVA myocytes, respectively (Table). Nevertheless, visual inspection of the records suggested that Kv current waveforms in Kvß₁^–/– and WT LVA myocytes are distinct. Kinetic analysis of the decay phases of the currents indeed revealed that I_to,f densities in Kvß₁^–/– LVA myocytes are significantly (P<0.01) lower than in WT LVA myocytes (Figure 4C). At +40 mV, for example, mean±SEM I_to,f densities were 25.3±1.4 and 21.0±0.9 pA/pF in WT and Kvß₁^–/– LVA myocytes, respectively. The voltage dependence of I_to,f activation and the kinetic properties of I_to,f in Kvß₁^–/– and WT LVA myocytes are similar (Table). Steady-state inactivation of I_to,f, however, is shifted by –10 mV in Kvß₁^–/– LVA myocytes (Table).

View larger version (29K): [in this window] [in a new window]   Figure 4. Ito,f and IK,slow densities are differentially affected in Kvß1–/– LVA myocytes. A, Whole-cell Kv currents were recorded from WT and Kvß1–/– LVA myocytes in response to 4.5 seconds depolarizing steps to test potentials between –40 to +50 mV (10-mV increments) from a holding potential of –70 mV. Currents were normalized for differences in cell size and current densities are presented. B, Mean±SEM peak Kv current densities in WT and Kvß1–/– myocytes are indistinguishable. Mean±SEM Ito,f densities (C) are significantly lower in Kvß1–/– LVA cells, whereas IK,slow densities (D) are significantly higher. E, Iss densities in Kvß1–/– and WT LVA myocytes are indistinguishable. (*P0.05, **P0.01)

View this table: [in this window] [in a new window]   Table 1. Properties of Voltage-Gated K+ Currents in WT and Kvß1–/– LVA Myocytes

Analysis of the Kv currents (Figure 4A) also revealed that mean±SEM I_K,slow density is significantly (P<0.01) higher in Kvß₁^–/–, than in WT LVA cells (Figure 4D). At +40 mV, for example, mean±SEM I_K,slow densities in Kvß₁^–/– and WT LVA cells were 19.1±0.9 and 15.9±0.7 pA/pF, respectively (Table). Is_s densities are similar in Kvß₁^–/– and WT LVA cells (Figure 4E and Table). Although I_K,slow densities are increased, the kinetics of I_K,slow inactivation and recovery from inactivation are similar in Kvß₁^–/– and WT LVA myocytes (Table). The voltage-dependence of I_K,slow inactivation, however, is also shifted (P0.05) by –10 mV in Kvß₁^–/–, compared with WT (n=12) LVA myocytes (Table).

Targeted Disruption of Kvß₁ Reduces I_to,f and Augments I_K,slow2
It has been demonstrated previously that Kv current components can be separated pharmacologically.^2,4–6,8 Mouse ventricular I_to,f, for example, is selectively reduced by nmol/L concentrations of Heteropodatoxin-2/3 (HpTx-2/3), whereas I_K,slow is attenuated preferentially by 4-AP and TEA.^5–8 Subsequent experiments, therefore, were focused on quantifying the effects of the targeted disruption of Kvß₁ on I_to,f and I_K,slow. Consistent with the kinetic analysis, the density of the 100 nmol/L HpTx-3–sensitive current is decreased significantly (P<0.01) in Kvß₁^–/– LVA myocytes (Figure 5A).

View larger version (14K): [in this window] [in a new window]   Figure 5. Pharmacological separation of Kv currents in WT and Kvß1–/– LVA myocytes. A, Outward Kv currents at +40 mV in Kvß1–/– and WT LVA myocytes were recorded as described in the legend to Figure 4 before, during, and after the application of 100 nmol/L HpTx-3 (left) or 50 µmol/L 4-AP and 5 mmol/L TEA (right). Application of 50 µmol/L 4-AP attenuates IK,slow1,5,6 and the subsequent application of 5 mmol/L TEA attenuates IK,slow2,7,8 as well as Iss.2 B, Densities and properties of the 100 nmol/L HpTx-sensitive Ito,f, the 50 µmol/L 4-AP–sensitive IK,slow1, and the 5 mmol/L TEA-sensitive IK,slow2 in individual cells were determined by analyzing the currents obtained by digital offline subtraction of the records in the absence and in the presence of the blockers. Mean±SEM density of the 100 nmol/L HpTx-sensitive current (Ito,f) is significantly (P<0.01) lower in Kvß1–/– (9.7±1.6 pA/pF; n=8), than in WT (12.9±1.7 pA/pF; n=10) LVA cells, whereas the density of the 5 mmol/L TEA-sensitive IK,slow2 is significantly higher in Kvß1–/– (5.9±0.4 pA/pF; n=12) than in WT (4.7±0.2 pA/pF; n=8) cells.

Previous studies have demonstrated that mouse ventricular I_K,slow reflects the expression of two molecularly distinct components. The µmol/L 4-AP–sensitive component of I_K,slow, I_K,slow1, is encoded by Kv1.5,^5,6 and the mmol/L TEA-sensitive component, I_K,slow2, is encoded by Kv2.1.^7,8 To determine whether I_K,slow1 and/or I_K,slow2 is affected by the targeted disruption of Kvß₁, the effects of 50 µmol/L 4-AP-and 5 mmol/L TEA were determined and compared. No significant differences in the densities of the 50 µmol/L 4-AP–sensitive I_K,slow1 were observed, whereas the density of the 5 mmol/L TEA-sensitive current, I_K,slow2, is significantly (P<0.01) higher in Kvß₁^–/– (n=12), than in WT (n=8) LVA myocytes (Figure 5B). Neither the voltage-dependences nor the kinetics of I_K,slow1 or I_K,slow2 activation or inactivation are measurably affected by the elimination of Kvß₁.

Similar experiments were completed on isolated myocytes from the septum of WT (n=12) and Kvß₁^–/– (n=8) hearts and identical results were obtained: I_to,f densities are reduced and I_K,slow2 densities are increased in Kvß₁^–/–, compared with WT, septum cells. Similar to the findings for the Kv1.5-encoded I_K,slow1 channels (Figure 5B), the densities and properties of the Kv1.4-encoded I_to,s channels are unaffected by the targeted disruption of Kvß₁.

Kvß₁/Kv Subunit Interactions in Adult Mouse Ventricles
In heterologous expression systems, it has been demonstrated that Kv and ß subunits associate.^17,19 To determine whether Kvß₁ subunits interact directly with Kv subunits in adult mouse ventricles, immunoprecipitations using a Kvß_1.1-specific monoclonal antibody were performed. As illustrated in Figure 6, the anti-Kvß_1.1 antibody reliably immunoprecipitates Kvß_1.1. Western blot analysis also revealed that Kvß_1.2, Kv4.2, and Kv4.3 coimmunoprecipitate with Kvß_1.1 (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 Kvß_1.1 (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 I_to,f channels. As would be expected, KChIP2 also coimmunoprecipitates with the anti-Kvß_1.1 antibody (online Figure OS1, available in the online data supplement).

View larger version (79K): [in this window] [in a new window]   Figure 6. Association between Kv subunits and Kvß1 in mouse ventricles. Homogenates prepared from adult mouse ventricles were immunoprecipitated (IP) using a monoclonal anti-Kvß1.1 antibody and immunoblotted (IB) with anti-Kvß1.1, anti-Kvß1.2, anti-Kv4.2, anti-Kv4.3, anti-Kv1.5 and anti-Kv2.1 antibodies. Protein preparations before IP were also blotted with the same (anti-Kv subunit) antibodies (lanes labeled IN). In addition to Kvß1.1, Kvß1.2, Kv4.2, and Kv4.3 coimmunoprecipitate with the monoclonal anti-Kvß1.1 antibody, and very little Kvß1.2, Kv4.3, or Kv4.3 remains in the supernatants (S). When the anti-Kvß1.1 antibody is omitted from the IP (–Ab), no precipitating Kv subunits are identified. Although both are readily detected in the supernatants, neither Kv1.5 nor Kv2.1 coimmunoprecipitates with anti-Kvß1.1.

In contrast to the findings with Kv4.2, Kv4.3, and KChIP2, neither Kv1.5 nor Kv2.1 coimmunoprecipitates with Kvß_1.1; both Kv1.5 and Kv2.1 are found only in the supernatants (Figure 6). Similar results were obtained using an anti–Kvß_1.2-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 Kvß_1.1 nor Kvß_1.2 was detected in the immunoprecipitated samples (not illustrated). In addition, immunoprecipitations with the anti-Kvß_1.1 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 Kvß_1.1 (online Figure OS2). Both Kv1.2, which is of unknown function, and Kv1.4, which encodes I_to,s,⁴ are found in the supernatants after immunoprecipitations with anti-Kvß_1.1 (online Figure OS2).

Targeted Disruption of Kvß₁ Alters the Membrane Expression of Kv Subunits
Similar to other accessory subunits,^11,12 Kvß subunits have chaperone-like effects, regulating the cell surface expression of Kv subunit–encoded 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 Kvß₁^–/– ventricles (Figure 7). Given that Kvß₁ subunits do not appear to interact directly with Kv2.1 (Figure 6), these results suggest that Kvß₁ exerts an indirect effect on mouse ventricular Kv2.1 expression. There are no significant differences in Kv1.5 membrane expression in WT and Kvß₁^–/– ventricles (Figure 7).

View larger version (35K): [in this window] [in a new window]   Figure 7. Membrane expression of Kv subunits is altered in Kvß1–/– ventricles. A, Western blots of fractionated adult WT and Kvß1–/– mouse ventricular membrane proteins were probed with the anti-Kv4.2, Kv4.3, Kv1.5, and Kv2.1 antibodies. B, Films from individual experiments were scanned, and the densities of the Kv4.2, Kv4.3, Kv2.1, and Kv1.5 bands in the Kvß1–/– and WT samples were measured and normalized. Mean±SEM expression of Kv4.3 is lower (P0.01) in Kvß1–/– (n=8) than in WT (n=8) ventricles, whereas no significant differences in Kv4.2 or Kv1.5 expression were seen. Kv2.1 expression, however, is higher (P0.001) in the Kvß1–/–, compared with WT, ventricles (n=8).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeted Disruption of Kvß₁ Attenuates Mouse Ventricular I_to,f
The results presented here demonstrate that I_to,f densities are markedly reduced in Kvß₁^–/– ventricular myocytes. The biochemical studies demonstrate that Kv4.2 and Kv4.3, as well as Kvß_1.2, coimmunoprecipitate with Kvß_1.1, and that the targeted disruption of Kvß₁ 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 I_to,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 Kvß_1.2,²⁰ Kvß3,²¹ MiRP1,²⁶ KChAP,²² DPPX,¹³ as well as by NCS1,²⁷ the voltage-gated Na⁺ (Nav) channel ß₁ subunit,²⁸ and the scaffolding protein, PSD-95.²⁹ Coexpression with Kvß₁ or Kvß₃, for example, increases the cell surface expression of Kv4.3-encoded channels,²¹ whereas coexpression with Kvß_1.2 modulates the properties, but not the expression, of Kv4.2-encoded channels.²⁰ In addition, biochemical studies have revealed that Kvß₁ subunits associate with Kv4 subunits in COS-1 cells,²¹ as well as in (rat) brain.³⁰ The results presented here suggest that Kvß₁ subunits contribute to the generation of mouse ventricular I_to,f channels by influencing the membrane expression of Kv4.3. The Kvß₁ 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 Kvß₁. 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 Kvß₁ subunits. It has also been reported, however, that Kvß₁ 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 Kvß_1.2, coimmunoprecipitate with Kvß_1.1, 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 I_to,f is also reduced in LVA myocytes isolated from mice in which only the N terminal Kvß_1.1 inactivation domain is removed, suggesting that the N-terminal domain of Kvß_1.1 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 Kvß₁ and Kv4 subunits and the roles of these interactions in controlling the functional cell surface expression of mouse ventricular I_to,f channels.

Targeted Deletion of Kvß₁ Augments Mouse Ventricular I_Kslow,2
Because Kv ß subunits have long been thought to interact specifically with subunits of the Kv1 subfamily,¹⁹ the working hypothesis at the outset here was that Kvß₁ likely associates with Kv1.5 and/or Kv1.4 and participates in the generation of mouse ventricular I_K,slow1 and/or I_to,s. No measurable effects on mouse ventricular I_K,slow1 or I_to,s, however, were evident in Kvß₁^–/– myocytes. In addition, the biochemical data suggest that (mouse) ventricular Kvß₁ does not associate with either Kv1.5 or Kv1.4. The results presented in this study, therefore, suggest that Kvß₁ subunits do not function as accessory proteins in the generation of mouse ventricular I_K,slow1 or I_to,s channels. The lack of association between Kv1.5 and Kvß_1.1 in adult mouse ventricles was unexpected given the previous findings that Kvß 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 Kvß₁ subunits, are clearly warranted to define the molecular basis of these disparate experimental observations.

Unexpectedly, the experiments here revealed that I_K,slow2 densities and the membrane expression of Kv2.1 are increased in Kvß₁^–/– ventricles. The biochemical data, however, do not suggest direct interaction(s) between Kv2.1 and Kvß_1.1 (or Kvß_1.2), at least in adult mouse ventricles. These results are consistent with previous studies that have failed to identify any interactions between Kvß₁ and Kv2.1 in (rat) ventricle²² or brain.³⁰ The possibility that Kvß₁ subunits might play an indirect role in the regulation of Kv2.1 channels, however, is suggested by the observation that Kvß_1.2 suppresses the modulatory effects of KChAP on Kv2.1-encoded K⁺ current expression in Xenopus oocytes.²² The upregulation of I_K,slow2 and Kv2.1 membrane expression demonstrated here suggest that endogenous Kvß₁ is a negative modulator of functional mouse ventricular I_K,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 I_K,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 rectifier^5,7,8 Kv currents in mouse ventricles is also associated with action potential and QT prolongation. The fact that action potential waveforms and QTc intervals in Kvß₁^–/– and WT myocytes/animals are indistinguishable likely reflects the fact that I_to,f and I_K,slow2 densities are differentially affected by the loss of Kvß₁, ie, the increase in I_K,slow2 appears to compensate for the decrease in I_to,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 Kvß_1.1 has been examined previously in studies on mice lacking Kvß_1.1 only (Kvß_1.1^–/–).³⁴ Phenotypic characterization of these mice suggested impaired learning and memory, and electrophysiological studies on Kvß_1.1^–/– 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, I_A, in isolated Kvß_1.1^–/– 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 I_A channels.^35,36 Interestingly, the decrease in I_A in Kvß_1.1^–/– CA1 neurons is accompanied by an increase in the sustained outward K⁺ current, I_SO,³⁴ which likely is encoded by Kv2.1.³⁷ It would clearly be interesting to determine whether the changes in I_A and I_SO densities reflect changes in the membrane expression of Kv4.x and/or Kv2.1 subunits in CA1 neurons.

The results presented here demonstrate that Kvß₁ participates in regulating the functional cell surface expression of native myocardial I_to,f and I_K,slow2 channels. Future experiments, using short interfering silencing RNAs (siRNAs) specific for Kvß_1.1 (or Kvß_1.2) in WT ventricular myocytes or expression of Kß_1.1 (or Kvß_1.2) in Kvß₁^–/– ventricular myocytes will provide insights into the specific role of these splice variants in the regulation of I_to,f and I_K,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 Kvß₁ subunits, the Kvß₁^–/– 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 Kvß₁^–/– mice and Drs Hullin Li and Kathryn Yamada for many helpful discussions.

	Footnotes

 
Original received July 22, 2004; revised resubmission received January 7, 2005; accepted January 12, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. 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: 25–61.

2. Xu H, Guo W, Nerbonne JM. Four kinetically distinct depolarization-activated K⁺ currents in adult mouse ventricular myocytes. J Gen Physiol. 1999; 113: 661–678.[Abstract/Free Full Text]

3. 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: 560–567.[Abstract/Free Full Text]

4. Guo W, Xu H, London B, Nerbonne JM. Molecular basis of transient outward K⁺ current diversity in mouse ventricular myocytes. J Physiol. 1999; 521: 587–599.[Abstract/Free Full Text]

5. 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 I_K,slow and resistance to drug induced QT prolongation. Circ Res. 2001; 88: 940–946.[Abstract/Free Full Text]

6. Li H, Guo W, Yamada KA, Nerbonne JM. Selective elimination of the 4-AP-sensitive component of delayed rectification, I_K,slow1, in mouse ventricular myocytes expressing a dominant negative Kv1.5 alpha subunit. Am J Physiol. 2004; 286: H319–H328.

7. Zhou J, Kodirov S, Murata M, Buckett PD, Nerbonne JM, Koren G. Regional upregulation of Kv2.1-encoded current, I_K,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice. Am J Physiol. 2003; 284: H491–H500.

8. 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: 623–633.[Abstract/Free Full Text]

9. 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: 357–398.[CrossRef][Medline] [Order article via Infotrieve]

10. 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: 344–355.[CrossRef][Medline] [Order article via Infotrieve]

11. 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: 11745–11751.[Abstract/Free Full Text]

12. 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: 553–556.[CrossRef][Medline] [Order article via Infotrieve]

13. 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: 449–461.[CrossRef][Medline] [Order article via Infotrieve]

14. 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: 289–294.[CrossRef][Medline] [Order article via Infotrieve]

15. 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: 6763–6767.[Abstract/Free Full Text]

16. Gulbis JM, Zhou M, Mann S, MacKinnon R. Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K⁺ channels. Science. 2000; 289: 123–127.[Abstract/Free Full Text]

17. 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: 843–852.[CrossRef][Medline] [Order article via Infotrieve]

18. Hoshi T, Zagotta WN, Aldrich RW. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990; 250: 533–538.[Abstract/Free Full Text]

19. Sewing S, Roeper J, Pongs O. Kv beta 1 subunit binding specific for Shaker-related potassium channel alpha subunits. Neuron. 1996; 16: 455–463.[CrossRef][Medline] [Order article via Infotrieve]

20. 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: 897–907.[Abstract/Free Full Text]

21. 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: 4839–4844.[Abstract/Free Full Text]

22. 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: C290–C299.

23. Deleted in proof.

24. 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: 586–593.[Abstract/Free Full Text]

25. 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: 103–120.[Abstract/Free Full Text]

26. 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: 1012–1019.[Abstract/Free Full Text]

27. 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: 26436–26443.[Abstract/Free Full Text]

28. Deschenes I, Tomaselli GF. Modulation of Kv4.3 current by accessory subunits. FEBS Lett. 2002; 528: 183–188.[CrossRef][Medline] [Order article via Infotrieve]

29. 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: 20423–20430.[Abstract/Free Full Text]

30. 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: 8246–8258.[Abstract/Free Full Text]

31. 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: 587–598.[CrossRef][Medline] [Order article via Infotrieve]

32. 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: 8736–8740.[Abstract/Free Full Text]

33. 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: H749–H759.

34. 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: 257–273.[Abstract/Free Full Text]

35. 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: 3124–3137.[Abstract/Free Full Text]

36. Malin S, Nerbonne JM. Elimination of the fast transient outward K⁺ current in superior cervical ganglion cells: molecular dissection of I_A. J Neurosci. 2000; 20: 5191–5199.[Abstract/Free Full Text]

37. 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: 69–81.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				C. V. DeSimone, Y. Lu, V. E. Bondarenko, and M. J. Morales S3b Amino Acid Substitutions and Ancillary Subunits Alter the Affinity of Heteropoda venatoria Toxin 2 for Kv4.3 Mol. Pharmacol., July 1, 2009; 76(1): 125 - 133. [Abstract] [Full Text] [PDF]

				D. Catalucci, D.-H. Zhang, J. DeSantiago, F. Aimond, G. Barbara, J. Chemin, D. Bonci, E. Picht, F. Rusconi, N. D. Dalton, et al. Akt regulates L-type Ca2+ channel activity by modulating Cav{alpha}1 protein stability J. Cell Biol., March 23, 2009; 184(6): 923 - 933. [Abstract] [Full Text] [PDF]

				Y.-J. Wang, B.-S. Chen, M.-W. Lin, A.-A. Lin, H. Peng, R. J. Sung, and S.-N. Wu Time-Dependent Block of Ultrarapid-Delayed Rectifier K+ Currents by Aconitine, a Potent Cardiotoxin, in Heart-Derived H9c2 Myoblasts and in Neonatal Rat Ventricular Myocytes Toxicol. Sci., December 1, 2008; 106(2): 454 - 463. [Abstract] [Full Text] [PDF]

				X. Li, K. Tang, B. Xie, S. Li, and G. J. Rozanski Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H416 - H424. [Abstract] [Full Text] [PDF]

				D. F. Steele, J. Eldstrom, and D. Fedida Mechanisms of cardiac potassium channel trafficking J. Physiol., July 1, 2007; 582(1): 17 - 26. [Abstract] [Full Text] [PDF]

				W. Guo, W. E. Jung, C. Marionneau, F. Aimond, H. Xu, K. A. Yamada, T. L. Schwarz, S. Demolombe, and J. M. Nerbonne Targeted Deletion of Kv4.2 Eliminates Ito,f and Results in Electrical and Molecular Remodeling, With No Evidence of Ventricular Hypertrophy or Myocardial Dysfunction Circ. Res., December 9, 2005; 97(12): 1342 - 1350. [Abstract] [Full Text] [PDF]

				J. M. Nerbonne and R. S. Kass Molecular Physiology of Cardiac Repolarization Physiol Rev, October 1, 2005; 85(4): 1205 - 1253. [Abstract] [Full Text] [PDF]

				M. D Ganfornina, M. T Perez-Garcia, G Gutierrez, E Miguel-Velado, J. R Lopez-Lopez, A Marin, D Sanchez, and C Gonzalez Comparative gene expression profile of mouse carotid body and adrenal medulla under physiological hypoxia J. Physiol., July 15, 2005; 566(2): 491 - 503. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 96/4/451    most recent 01.RES.0000156890.25876.63v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aimond, F. Articles by Nerbonne, J. M. Search for Related Content PubMed PubMed Citation Articles by Aimond, F. Articles by Nerbonne, J. M. Related Collections Other myocardial biology Animal models of human disease Arrythmias-basic studies