Kvbeta Subunits Increase Expression of Kv4.3 Channels by Interacting with Their C Termini*

Eun-Kyoung Yang, Mauricio R. Alvira, Edwin S. LevitanDagger, and Koichi Takimoto§

From the Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, June 1, 2000, and in revised form, November 6, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Auxiliary Kvbeta subunits form complexes with Kv1 family voltage-gated K+ channels by binding to a part of the N terminus of channel polypeptide. This association influences expression and gating of these channels. Here we show that Kv4.3 proteins are associated with Kvbeta 2 subunits in the brain. Expression of Kvbeta 1 or Kvbeta 2 subunits does not affect Kv4.3 channel gating but increases current density and protein expression. The increase in Kv4.3 protein is larger at longer times after transfection, suggesting that Kvbeta -associated channel proteins are more stable than those without the auxiliary subunits. This association between Kv4.3 and Kvbeta subunits requires the C terminus but not the N terminus of the channel polypeptide. Thus, Kvbeta subunits utilize diverse molecular interactions to stimulate the expression of Kv channels from different families.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Voltage-gated K+ channels are multimeric proteins that consist of four pore-forming alpha  subunits in association with auxiliary subunits. Unlike alpha  subunits that exhibit overall structural similarity to various voltage-gated ion channels, auxiliary subunits are diverse and show specificity for association with particular classes of K+ channels. Kvbeta subunits belong to the NAD(P)H-dependent oxidoreductase superfamily (1) and contain a conserved catalytic domain with a NADPH-binding site (2). These proteins by themselves form a tetramer (3), making an alpha 4beta 4 channel complex (4-6). There are at least four mammalian genes that encode Kvbeta subunits. Previous studies have established that Kvbeta 1, -2, and -3 gene products are components of Kv1 family channels. Indeed, all products from the four genes contain a conserved core region with variable N-terminal peptides. The core region of beta  subunits was found to be sufficient for association with a part of the N terminus of Kv1 family alpha  subunits that is highly conserved within this family (5, 6). However, more recent studies indicate that Kvbeta subunits can interact with heterologously expressed Kv4.2 (7, 8) and EAG1 family (9) channels. Furthermore, it appeared that Kvbeta 4 subunits are present as complexes with Kv2.2 proteins in rat brain (10). Likewise, plant Kvbeta subunits (KAB) are associated with KAT1 channels (11). Hence, auxiliary Kvbeta subunits are structurally well characterized, yet the specificity and mechanism of interaction between Kvalpha and beta  subunits remain obscure.

Kvbeta subunits influence expression and function of K+ channels. Specifically, distinct Kvbeta subunits differentially affect heterologously expressed Kv1 family channels. A long-stretched N-terminal peptide in Kvbeta 1 and Kvbeta 3 gene products produces rapid inactivation on most of Kv1 family channels by a mechanism similar to the action of a ball peptide present at the N terminus of some channel alpha  subunits (12). Furthermore, Kvbeta 2 subunits have been shown to increase stability and cell surface expression of Kv1 family channels (13) without producing rapid inactivation. Thus, expression of distinct Kvbeta subunits controls excitability by differentially affecting the expression and gating of Kv1 family channels.

Although many studies have shown that Kvbeta 1 and Kvbeta 2 subunits can associate with heterologously expressed channels from diverse families, it remains unclear whether these auxiliary subunits are present as complexes with non-Kv1 family channels in native cells. Furthermore, structural features of the interaction between Kvbeta subunits and non-Kv1 family channel polypeptides remain unknown. To address these questions, we examined complexes consisting of Kvbeta and Kv4.3 channel subunits. We show here that Kv4.3 proteins are associated with Kvbeta 2 subunits in the brain and that this interaction requires the C terminus of the channel polypeptide.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constructions-- Rat Kv4.3 short (14) and long (15) isoform cDNAs were subcloned into pcDNA3 (Invitrogen, Carlsbad CA). Rat Kv2.1, beta 1.1. and beta 2.1 expression constructs were previously obtained (7). Chimeric constructs between Kv4.3 short splicing form and Kv2.1 were made using a two-step overlapped polymerase chain reaction with primers that corresponded to the border region of Kv4.3 and Kv2.1 sequences. Kv4.3-Kv2.1N contains amino acids 1-184 of rat Kv2.1 polypeptide (16) linked to amino acids 183-636 of rat Kv4.3 polypeptide (14). Kv4.3-Kv2.1C consists of amino acids 1-406 of the Kv4.3 polypeptide connected to amino acids 413-853 of the Kv2.1 polypeptide. For coisolation experiments, wild type and chimeric channel cDNAs were subcloned in-frame into pcDNA3.1/HisC vector (Invitrogen) using a polymerase chain reaction-based method. GFP-tagged Kvbeta subunit constructs were prepared by subcloning the whole coding region of Kvbeta 1.1 and Kvbeta 2.1 cDNAs at the end of enhanced green fluorescent protein-coding sequence of EGFP-C1 (CLONTECH, Palo Alto CA). All the obtained constructs were verified by DNA sequencing.

Cell Culture and Transfection-- HEK 293 cells (American Type Culture Collection, Manassas, VA) were maintained at 37 °C under 5% CO2 atmosphere in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transient transfection was carried out by the calcium phosphate-DNA coprecipitation method (Transfinity, Life Technologies, Inc.).

For patch clamp recording, pCMV-Kv4.3 alone (0.3 µg for 60-mm dish) or in combination with 5×-excess pCMV-Kvbeta 1.1 or pCMV-Kvbeta 2.1 were used. In addition, EGFP-C1 plasmid (50 ng/60-mm dish) was cotransfected to aid in the identification of transfected cells. Transfected cells in 60-mm plates were split into 35-mm dishes 5 h after transfection and used for whole-cell recordings 48-72 h after transfection.

For immunoblot analysis, cells on 100-mm plates were transfected with expression constructs at the same ratio as for patch clamp recording (0.9 µg of pCMV-Kv4.3/dish). Transfected cells were divided into five 60-mm plates at various densities 5 h after transfection and used for immunoblot analysis at various days after transfection. Cell extracts were prepared by suspending the collected cell pellet in 100 µl of lysis buffer (20 mM Tris-HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 1 mM iodoacetamide, 0.2 mM phenylmethylsulfoxide, and 1 mM EDTA). The suspension was kept on ice for 10 min and centrifuged at 10,000 × g for 5 min to remove nuclear debris.

Electrophysiological Recording-- Whole-cell voltage-clamp recording (17) was performed with an EPC-9 patch-clamp amplifier using the Pulse program (HEKA Electronik, Lambrecht, Germany) on a Power Macintosh computer. Patch pipettes were filled with a solution containing 140 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM HEPES (pH 7.4). Bath solution contained 155 mM NaCl, 5 mM KCl, 2 mM MgCl2, 20 mM glucose, 10 mM HEPES (pH 7.4). Series resistance compensation was set at 70%. Peak currents were converted into conductance (G) by the formula G = I/(Vm - Vrev) assuming a reversal potential Vrev of -84 mV, where Vm is the membrane voltage of depolarization pulses. Using the first-order Boltzmann equation G/Gmax = 1/(1 + exp[(V1/2 - V)/slope factor]), the half-maximal voltages (V1/2) and the slope factors were acquired. Statistical analysis was carried out using the Mann-Whitney two-tailed test. All the data in the text are presented as means ± S.E.

Biochemical Association Assays-- Two days after transfection, transfected HEK 293 cells on 100-mm dishes were harvested with ice-cold phosphate-buffered saline. Triton extract was prepared by suspending the pelleted cells in 0.4 ml of solution containing 1% Triton X-100, 20 mM Tris-HCl (pH 7.9), 50 mM NaCl, and 5 mM imidazole. The extract was mixed with 100 µl (50% slurry) of preactivated His-bind resin (Novagen, Milwaukee WI) for 2 h with gentle shaking. The resin was washed 5× with the same solution, except that the imidazole concentration was 40 mM. The bound materials were then eluted with 0.1 M EDTA.

Immunoprecipitation-- Immunoprecipitation was performed with polyclonal anti-panKvbeta antibody (18) or monoclonal anti-Kv4.3 antibody. The latter antibody was generated against a synthetic peptide corresponding to a part of the N terminus of rat Kv4.3 polypeptide (amino acids 25-40) CPMPLAPADKNKRQDE.2

Whole rat brain tissue was homogenized in 0.32 M sucrose solution supplemented with 1 mM iodoacetamide, 0.2 mM phenylmethyl sulfonate, and 1 mM EDTA. The homogenate was centrifuged at 1,000 × g for 10 min to remove nuclear debris. The supernatant was transferred to a new tube and centrifuged at 100,000 × g for 1 h. Protein concentration was determined using Bio-Rad protein assay reagent with bovine serum albumin as a standard. The nuclei-free membrane fraction was suspended in a solution containing 2% Triton X-100, 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride and 1 mM iodoacetamide at a protein concentration of ~10 mg/ml. Triton extract was then obtained by centrifugation of the suspension at 100,000 × g for 30 min. After preclearing with fixed protein A-containing Staphylococcus aureus cells (Pansorbin, Calbiochem), Triton extract was incubated overnight with monoclonal anti-Kv4.3 or polyclonal anti-pan Kvbeta (20) antibody and Pansorbin. The bound materials were collected by centrifugation and washed 4× with the same Triton-containing solution. The bound materials were eluted by heating in 2× SDS sample buffer and subjected to immunoblot analysis.

Immunoblot Analysis-- Proteins were separated on a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was coated with 5% nonfat dry milk in phosphate-buffered saline containing 0.1% Tween 20 and probed with primary antibody followed by incubation with horseradish peroxidase-conjugated secondary antibody. Primary antibodies against Kv4.3, GFP, and Express tag were purchased from Alomone labs (Jerusalem, Israel), MBL International Corp. (Watertown MA), and Invitrogen, respectively. Anti-Kv1.4 (18), anti-Kv2.1 (19), and polyclonal anti-panKvbeta (20) antibodies were previously generated. Bound antibody was detected by chemiluminescence method (PerkinElmer Life Sciences). Immunoreactivity was quantified using densitometry of the developed films.

Confocal Microscopy-- Confocal Images of GFP fluorescence were taken on a Molecular Dynamic 2001 scanning laser confocal microscope with a 60× oil immersion objective lens (1.4 NA) using 488-nm excitation and 510-nm emission filters with 3% maximal laser intensity. Cell surface localization was evaluated by comparing the location of fluorescence with bright field images of cells.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kv4.3 Proteins Are Present in Association with Kvbeta 2 Subunits in the Brain-- To test for the presence of Kv4.3·Kvbeta subunit complexes, we first used anti-Kv4.3 monoclonal antibody for immunoprecipitation from rat brain extract. Anti-Kv4.3 antibody effectively and specifically precipitated its targeted channel proteins but not Kv1.4 proteins (Fig. 1A). Importantly, the immunoprecipitated material was found to contain significant immunoreactive Kvbeta subunit proteins detected with polyclonal anti-panKvbeta antibody (20). This antibody detects two bands on the blot with distinct sizes. The larger and smaller bands are known to correspond to Kvbeta 1 and Kvbeta 2, respectively (13, 19, 20). In addition, the larger band may also contain Kvbeta 3 subunits. We found that only smaller molecular weight Kvbeta 2 subunits were significant in the precipitated material. To further obtain evidence for the presence of Kv4.3·Kvbeta complexes in the brain, anti-panKvbeta antibody was used for immunoprecipitation from the brain extract (Fig. 1B). The antibody precipitated significant Kv4.3 proteins in addition to its targeted proteins. In contrast, no detectable Kv2.1 proteins were found in the precipitated material. Hence, brain Kv4.3 channel proteins are present in association with Kvbeta 2 subunits.



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Fig. 1.   Kv4.3 proteins are associated with Kvbeta 2 subunits in rat brain. Triton extract of whole rat brain was subjected to immunoprecipitation (IP) with monoclonal anti-Kv4.3 (A) or polyclonal anti-panKvbeta (B) antibody. Brain extract, unbound fraction (sup), and precipitated material (ppt) were examined by immunoblot (IB) analysis with anti-Kv1.4, anti-Kv2.1, anti-panKvbeta , and polyclonal anti-Kv4.3 antibodies. The larger and smaller bands detected with anti-panKvbeta antibody have been shown to correspond to Kvbeta 1/3 and Kvbeta 2 subunits, respectively (13, 19, 20). Note that a fraction of immunoreactive Kvbeta subunits (Kvbeta 2) is recovered in the anti-Kv4.3 antibody-precipitated material. Anti-panKvbeta antibody also precipitated a portion of Kv4.3 proteins.

Kvbeta Subunits Increase Kv4.3 Current Density and Proteins-- Functional consequences of Kvbeta subunit association on Kv4.3 channels was examined in transfected HEK 293 cells. We first examined the effect of Kvbeta subunits on Kv4.3 current density. Coexpression of either Kvbeta 1.1 or Kvbeta 2.1 subunits led to a 2-3-fold increase in the peak current density (Fig. 2); at +50 mV, peak current density was 0.5 ± 0.05 nA/pF (n = 6) for Kv4.3 alone, 1.7 ± 0.4 nA/pF (n = 5) for Kv4.3 + Kvbeta 1.1, and 1.8 ± 0.5 nA/pF (n = 5) for Kv4.3 + Kvbeta 2.1. Increases in current density produced by Kvbeta 1.1 or Kvbeta 2.1 subunits were significant at test pulses higher than -20 mV (p < 0.05). No significant change in HEK 293 cell endogenous current was detected; peak current density at +50 mV was 39.2 ± 8.7 pA/pF (n = 4) for mock transfection, 29.3 ± 9.9 pA/pF (n = 4) for Kvbeta 1.1, and 39.7 ± 7.1 pA/pF (n = 4) for Kvbeta 2.1. Thus, Kvbeta subunits increase functional cell surface Kv4.3 channels.



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Fig. 2.   Kvbeta subunits increase Kv4.3 channel current density. HEK 293 cells were transfected with expression constructs for Kv4.3 alone, Kv4.3 + Kvbeta 1.1 or Kv4.3 + Kvbeta 2.1 (alpha :beta  = 1:5). Outward currents were elicited by 200-ms depolarization pulses with 10-mV increments from a holding potential of -70 mV. Current density was obtained with cellular capacitance to account for deviation in cell size. A, representative current traces are shown. Scale bars: horizontal, 50 ms; vertical, 250 pA/pF. B, current-voltage relationships are presented. Points and error bars represent mean and S.E., respectively. n >=  4 for each transfection condition. Current density is significantly larger with expression of Kv4.3 + Kvbeta 1.1 or Kv4.3 + Kvbeta 2.1 than that of Kv4.3 alone at a membrane depolarization above -20 mV (p < 0.05).

To test if this elevation in current density was correlated with an increase in Kv4.3 protein level, we measured channel proteins by immunoblot analysis (Fig. 3A). The Kv4.3 protein level significantly increased when coexpressed with Kvbeta 2.1 subunits 3 or 4 days after transfection (Fig. 3B, n = 6, p < 0.05). In contrast, coexpression of these auxiliary subunits did not produce changes in Kv2.1 protein levels. Similar increases in Kv4.3 proteins were also produced by Kvbeta 1.1 at 3 days after transfection (Fig. 3C). Hence, Kvbeta subunits increase total cellular Kv4.3 protein level.



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Fig. 3.   Kvbeta subunits increase Kv4.3 channel protein level. HEK 293 cells were transfected with expression constructs for Kv4.3 or Kv2.1 channels with Kvbeta 2.1 or control vector (alpha :beta  = 1:5). Various days after transfection, cells were harvested for immunoblot analysis. A, immunoblots with anti-Kv4.3 antibody (top) and anti-Kv2.1 antibody (bottom) are shown. B, densitometry was used to quantify immunoreactivity. Kv4.3 (top) and Kv2.1 (bottom) immunoreactivities were normalized with total protein amounts. n = 6 for each time point. Points and error bars represent mean and S.E., respectively. An asterisk indicates p < 0.05. C, cells were transfected expression constructs for Kv4.3 channel proteins with Kvbeta 1.1, Kvbeta 2.1, or control vector. Three days after transfection, cell extracts were prepared, and channel immunoreactivities were measured. An asterisk indicates p < 0.05. w/o, without.

Effects of Kvbeta Subunits on Kv4.3 Channel Gating-- Next we examined the gating properties of Kv4.3 channels with or without coexpression of Kvbeta subunits. Excess Kvbeta subunits (alpha :beta  = 1:5) were used to enhance formation of Kv4.3·Kvbeta complexes. Kvbeta subunits have been shown to shift voltage dependence of activation of Kv1 family channels to the left. However, we found that Kvbeta 1.1 or Kvbeta 2.1 subunits produce no significant change in voltage dependence of activation of Kv4.3 channels (Fig. 4A). The voltage for half-maximal activation and the slope factor were -7.00 ± 1.08 mV and 14.0 ± 0.99 mV (n = 5) for Kv4.3 alone, -7.83 ± 1.01 mV and 15.8 ± 0.96 mV (n = 4) for Kv4.3 + Kvbeta 1.1, and -8.43 ± 1.13 mV and 14.4 ± 1.05 mV (n = 4) for Kv4.3 + Kvbeta 2.1, respectively. Thus, Kvbeta subunits do not influence activation of Kv4.3 channels.



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Fig. 4.   Kvbeta subunits produce no marked effects on Kv4.3 channel gating. Outward currents were measured in HEK 293 cells transfected with expression constructs for Kv4.3 alone, Kv4.3 + Kvbeta 1.1, or Kv4.3 + Kvbeta 2.1 (alpha :beta  = 1:5). Conductance-voltage relation (A) was obtained using pulse paradigm shown in Fig. 2. Currents at 50-mV depolarization pulses were normalized, and the averages of these traces (n >=  4) are shown in B. Steady-state inactivation (D) and recovery from inactivation (F) were determined using pulse paradigms shown in C and E, respectively. n >=  4 for each point. Points and error bars represent mean and S.E., respectively. No significant differences in any of the three gating properties between any two conditions were detected.

The most profound effect produced by Kvbeta subunits on Kv1 family channels is acceleration of inactivation, due to the ball and chain mechanism. Thus, the effect of Kvbeta 1.1, which contains a ball peptide, as well as Kvbeta 2.1 on inactivation properties of Kv4.3 channels was examined. Coexpression of these auxiliary subunits did not significantly affect time constant of inactivation (Fig. 4B); time constant for inactivation at +50 mV was 50.5 ± 5.1 ms for Kv4.3 alone (n = 5), 52.6 ± 4.3 ms (n = 4) for Kv4.3 + Kvbeta 1.1, and 57.2 ± 2.2 ms (n = 4) for Kv4.3 + Kvbeta 2.1. We also measured the steady-state inactivation using a test pulse to +40 mV after a conditioning prepulse at various voltages (Fig. 4, C and D). The voltage for half-maximal inactivation and the slope factor were -48.4 ± 0.33 mV and -5.2 ± 0.3 mV (n = 5) for Kv4.3 alone, -41.5 ± 0.23 mV and -6.8 ± 0.2 mV (n = 4) for Kv4.3 + Kvbeta 1.1, and -47.2 ± 0.45 mV and -6.85 ± 0.4 mV (n = 4) for Kv4.3 + Kvbeta 2.1. Therefore, Kvbeta subunits, regardless of the presence of a ball peptide, do not influence inactivation of Kv4.3 channels.

We also determined whether association with Kvbeta 1.1 or Kvbeta 2.1 might influence the recovery from inactivation (Fig. 4, E and F). A protocol of two consecutive depolarizing test pulses interrupted by variable interpulse intervals at -70 mV was used to determine the time course of recovery from inactivation (Fig. 4E). The recovery from inactivation was fitted by a single exponential function. Time constant for recovery from inactivation was 181 ± 24 ms for Kv4.3 alone (n = 6), 169 ± 11 ms for Kv4.3 + Kvbeta 1.1 (n = 5), and 176 ± 38 ms for Kv4.3 + Kvbeta 2.1 (Fig. 4F, n = 5). Thus, Kvbeta subunits do not affect recovery from inactivation. Taken together, Kvbeta 1.1 and Kvbeta 2.1 subunits produce no marked effects on Kv4.3 channel gating.

The C Terminus of Kv4.3 Proteins Is Required for Association with Kvbeta Subunits-- To assess association of Kv channel alpha  subunits with Kvbeta subunits, we first examined localization of GFP-tagged Kvbeta subunits upon coexpression of various channel proteins. Confocal microscopy revealed that GFP-Kvbeta 2.1 (Fig. 5A) or GFP-Kvbeta 1.1 (data not shown) were predominantly present in the cytosol in the absence of channel alpha  subunits. Coexpression of Kv4.2 or Kv4.3 proteins as well as Kv1.4 proteins, but not Kv2.1 proteins, localized the fluorescence to plasma membrane (Fig. 5A). Similarly, a splicing variant of Kv4.3, which contains a 19-amino acid insertion at the C terminus (15), targeted GFP-Kvbeta fusion proteins to plasma membrane. Thus, Kv4 family channel proteins regardless of the presence or absence of the insertion can associate with Kvbeta subunits.



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Fig. 5.   The C terminus of Kv4.3 is required for localizing GFP-tagged Kvbeta subunits to plasma membrane. A, HEK 293 cells were transfected with expression constructs for GFP-tagged Kvbeta 2.1 and indicated Kv channel alpha  subunits at the ratio of alpha :beta  = 5:1. Kv4.3S and Kv4.3L represent short and long splicing isoforms of Kv4.3alpha subunits, respectively. Confocal images were obtained 2 days after transfection. Coexpression of Kv4.2, Kv4.3S, Kv4.3L, or Kv1.4 alters the location of the fluorescence to cell surface (a ring-like structure), whereas the fluorescence is predominantly seen in cytosol with GFP-Kvbeta 2.1 alone or GFP-Kvbeta 2.1 + Kv2.1. B, wild type or chimeric channel alpha  subunits were expressed with GFP-Kvbeta 2.1 fusion proteins into HEK 293 cells at the expression construct ratio of alpha :beta  = 5:1. Confocal images 2 days after transfection are shown. Kv4.3-2.1N and Kv4.3-2.1C indicate chimeric Kv4.3 proteins containing the N terminus and C terminus of Kv2.1 polypeptide instead of their own, respectively. Note that replacing the C terminus, but not the N terminus, eliminated the ability of Kv4.3 proteins to transfer GFP-Kvbeta subunits to plasma membrane.

Kv1 family channels interact with Kvbeta subunits via a highly conserved region of the N terminus. Although the corresponding region of Kv4 family polypeptides exhibits significant sequence homology, this peptide itself was insufficient for association with Kvbeta subunits (5, 6). To identify the region important for association, we generated chimeric channel proteins consisting of Kv4.3 and Kv2.1 polypeptides. If chimeric proteins are capable of interacting with Kvbeta subunits, the fluorescence would be expected in plasma membrane or other membrane-associated compartments. Replacing the N terminus of Kv4.3 protein with that of Kv2.1 polypeptide (Kv4.3-Kv2.1N) did not affect the ability to localize Kvbeta 2.1 (Fig. 5B) and Kvbeta 1.1 (data not shown) subunits to plasma membrane and other membrane-associated regions. In contrast, substituting the C terminus of Kv4.3 protein with that of Kv2.1 protein (Kv4.3-Kv2.1C) eliminated plasma membrane localization of the fluorescence. This chimeric channel (Kv4.3-Kv2.1C) was functional as confirmed by patch clamp recording (data not shown). Thus, the C terminus, but not the N terminus, of Kv4.3 polypeptide is required for localizing Kvbeta subunits at plasma membrane.

We also used protein biochemical assays to test association. Histidine (His6)-tagged wild type and chimeric channel proteins were expressed with Kvbeta 1.1 or Kvbeta 2.1 subunits. After purification with His-binding beads, copurified Kvbeta subunit proteins were examined by immunoblot analysis (Fig. 6). Significantly higher levels of immunoreactive Kvbeta 1.1 or Kvbeta 2.1 proteins were recovered from cells coexpressed with Kv4.3 or Kv4.3-Kv2.1N than those with Kv2.1 or Kv4.3-Kv2.1C. These results demonstrate that the C terminus, but not the N terminus, of Kv4.3 channels is necessary for association with Kvbeta subunits.



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Fig. 6.   The C terminus of Kv4.3 proteins is necessary for association with Kvbeta subunits. (His)6- and Express epitope-tagged wild type and chimeric alpha  subunits were expressed with Kvbeta 1.1 (left) or Kvbeta 2.1 (right) subunits into HEK 293 cells at the expression construct ratio of alpha :beta  = 1:1. Two days after transfection, cell lysate was prepared and subjected to isolation with His-bind beads. Input and ppt represent immunoblots obtained with cell lysate and His-bind bead-bound fraction, respectively. A small amount of Kvbeta subunit proteins similar to the Kv2.1 and Kv4.3-2.1C lanes was also obtained without expression of any channel alpha  subunits.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kvbeta subunits have been suggested to interact with various K+ channels including Kv2.2 (10), Kv4.2 (7, 8), and several EAG family (9) and plant KAT1 (11) channels in addition to Kv1 family channels. In particular, Kv4.2 channels were found to interact with Kvbeta 1 and -2 subunits in heterologous expression systems (7, 8), suggesting that the same Kvbeta subunits might form complexes with K+ channels from the two different families. However, previous studies had not addressed whether such association actually occurs with native channels and did not identify a physiological effect of the association. In this study, we have shown that Kv4.3 proteins are associated with Kvbeta 2 subunits in the brain. We also found that coexpression of Kvbeta subunits leads to increases in Kv4.3 current density and protein level without altering gating properties. Finally, this association requires the C terminus, but not the N terminus, of the channel polypeptide. Thus, the same Kvbeta 2 subunits influence expression and function of channels from the two different families by distinct interaction mechanisms.

Association of Kvbeta subunits appeared to produce different effects on interacting channels. Our results indicate that the ball peptide of Kvbeta 1.1 does not alter inactivation of Kv4.3 channels. Similarly, it has been shown that Kvbeta 1 does not markedly influence inactivation kinetics of Kv4.1 (21), Kv4.2, (8) and Drosophila Shal (5) channels. Likewise, Drosophila Kvbeta subunit HK was unable to produce rapid inactivation on EAG or ERG channels (9). Thus, the effect of Kvbeta subunit ball peptide depends on associating channels. The lack of the ball peptide effect may be due to differences in the ball peptide-accepting structure of channels. Recent work indicated that a difference in the amino acid sequence in a small region of the linker between S4 and S5 affects the interaction with the Kvbeta 1 ball peptide (22). To support this possibility, one of the important amino acids identified in the above-mentioned study, Arg-324 in Kv1.1, is altered to serine at the corresponding position (Ser-421) in Kv4.3. Another possibility is that Kvbeta subunits are positioned in such a way that the ball peptide cannot access the internal pore region of Kv4.3 channels. Since we found that structural requirement for association with Kvbeta subunits differs between Kv1 family and Kv4.3 channels, it is possible that the relation of a Kvbeta tetramer to a Kv4.3alpha tetramer does not allow the ball peptide to act on its receptor region of the channel. In addition to the difference in rapid inactivation, a recent study revealed that sensitivity to O2 tension differs between Kvbeta subunit-associated Kv4.2 and Shaker channels (8). In analogy to the difference in rapid inactivation, this difference in hypoxia response may arise from distinct ability of channels to respond O2 tension signals. Alternatively, specific interaction of Kv4.2 channels with Kvbeta subunits may be essential for the regulation. Further structural and functional information of channel complexes may resolve these issues.

In contrast to specific alterations in channel gating, association of Kvbeta subunits commonly increases current amplitude or density of various channels. This has been observed with Kv1 family (13), Kv2.2 (10), and EAG family (9) channels. Our results also revealed that Kvbeta subunits increase Kv4.3 current density and proteins. In addition to Kvbeta subunits, other channel auxiliary subunits for K+ channels as well as Na+ and Ca2+ channels have been shown to increase associating channel current density. It is assumed that the exit from endoplasmic reticulum is the rate-limiting step for plasma membrane protein targeting. Therefore, the generally observed increase in current density by various auxiliary subunits may be due to masking of endoplasmic reticulum retention signals present in channel proteins. This mechanism has been implicated for controlling selective cell surface expression of heteromeric ATP-sensitive K+ channel complexes (23, 24) and voltage-gated Ca2+ channel (25). Thus, it is possible that some of the Kvbeta subunit effect on Kv4.3 channel current density and proteins may be due to masking potential endoplasmic reticulum retention signals in the channel polypeptide. However, our previous study found that GFP-tagged Kv1.4 and Kv1.5 channels are efficiently transported to plasma membrane in the absence of Kvbeta subunits (26, 27). Similarly, we found efficient plasma membrane localization of GFP-tagged Kv4.3 (data not shown). Furthermore, coexpression of Kvbeta subunits produced no apparent changes in localization of these GFP-tagged channel proteins. Thus, it is likely that Kvbeta subunits increase Kv4.3 as well as Kv1 family proteins in endoplasmic reticulum and at the plasma membrane. This stabilization effect is further supported by our finding that the Kvbeta effect on Kv4.3 protein level is larger at longer times after transfection. Hence, Kv4.3·Kvbeta complexes are likely more stable than those without these auxiliary subunits.

Despite the similarity between Kv4 and Kv1 family polypeptides, our data indicate that the two family proteins exhibit distinct requirements for interaction with Kvbeta subunits. A part of the N terminus of Kv1 family polypeptide is sufficient for association (5, 6). In contrast, our results demonstrated that the corresponding region of Kv4 family peptide is not necessary. Instead, the association requires the C terminus of Kv4.3 polypeptide. The importance of the C terminus for interaction with Kvbeta subunits was also suggested in Kv2.2·Kvbeta 4 complex formation; a part of the C terminus of Kv2.2 protein is required for the increase in current density produced by Kvbeta 4 coexpression in Xenopus oocytes (10). Thus, the C terminus of Kv2.2 and Kv4 family polypeptides is likely to be involved in association with Kvbeta subunits. The apparent lack of sequence similarity between the N terminus of Kv1 family and the C termini of Kv2.2 or Kv4.3 polypeptides suggest that the interaction between Kvalpha and -beta subunits may be more complex than previously assumed. To further elucidate interaction mechanisms, we generated a chimeric Kv2.1 channel containing the C terminus of Kv4.3 polypeptide. We found that this chimera does not efficiently associate with Kvbeta subunits (data not shown), suggesting that the Kv4.3 C terminus may not be sufficient for association. However, this chimera was found to be nonfunctional. Therefore, misfolding of this chimeric channel protein might be responsible for the observed lack of interaction. Thus, a simple explanation for the requirement of the C terminus is that this peptide interacts with a site of Kvbeta polypeptide that is distinct from one for the Kv1 family N terminus. Alternatively, the C terminus may indirectly participate in interaction. For example, the C-terminal peptide interacts with other part of the channel protein to place an association site in a position for efficient interaction with these auxiliary subunits. More detailed analyses are required to differentiate these possibilities.

Recently identified Ca2+-binding subunits (KChIP) are likely to play important roles in controlling the expression and function of Kv4 family channels (28). In addition, our results indicate that Kv4.3 channels are present, at least in part, in association with Kvbeta 2 subunits in brain. The association of non-Kv1 family channels with Kvbeta subunits may have pronounced effects under physiological conditions. Although mutations in Drosophila Kvbeta subunit HK and Kv1 channel Shaker resulted in almost identical electrophysiological changes in some giant neurons, alterations in other types of neurons caused by these HK mutations were distinct from those by Shaker mutations (29). Thus, the interaction of Kvbeta subunits with non-Kv1 channels is likely to play important roles in controlling neuronal excitability. Our results indicate that the major effect produced by Kvbeta subunits on Kv4 family channels is to increase the number of functional channels. The association of Kvbeta subunits may also have other regulatory functions on these channels, such as sensing redox state of the cell. A novel interaction mechanism between Kv4 family channels and Kvbeta subunits may be important for these regulatory functions. Hence, Kvbeta subunits control neuronal excitability by influencing expression and function of Kv1 and Kv4 family channels.


    ACKNOWLEDGEMENTS

We thank Drs. J. E. Dixon and D. McKinnon for rat Kv4.3 cDNA and Dr. J. S. Trimmer for rat Kvbeta 1.1 and Kvbeta 2.1 cDNAs and anti-Kv4.3, anti-Kv2.1, and anti-panKvbeta antibodies.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL55312 (to E. S. L.) and HL63123 (to K. T.), a grant-in-aid from the American Heart Association Pennsylvania affiliate (to K. T.), and a postdoctoral fellowship from the Korean Science and Engineering Foundation (to E.-K. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger An Established Investigator of the American Heart Association.

§ To whom correspondence should be addressed: E1355 Biomedical Science Tower, Dept. of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-383-7325; Fax: 412-648-1945; E-mail: koichi+@pitt.edu.

Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M004768200

2 J. S. Trimmer, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: EAG, ether-a-gogo; GFP, green fluorescent protein; pF, picofarads; HEK, human embryonic kidney.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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