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Originally published In Press as doi:10.1074/jbc.M400069200 on January 30, 2004

J. Biol. Chem., Vol. 279, Issue 15, 15531-15540, April 9, 2004
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Role of the NH2 Terminus in the Assembly and Trafficking of the Intermediate Conductance Ca2+-activated K+ Channel hIK1*

Heather M. Jones{ddagger}, Kirk L. Hamilton§, Glenn D. Papworth{ddagger}, Colin A. Syme{ddagger}, Simon C. Watkins{ddagger}, Neil A. Bradbury{ddagger}, and Daniel C. Devor{ddagger}

From the {ddagger}Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the §Department of Physiology, University of Otago, Dunedin, New Zealand

Received for publication, January 5, 2004 , and in revised form, January 29, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of the NH2-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH2-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 ± 1.3 min compared with 3.2 ± 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH2 terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 °C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH2 terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The KCNN gene family is composed of four members, including the small conductance Ca2+-activated K+ channels (SK1, SK2, and SK3)1 and the intermediate conductance Ca2+-activated K+ channel (IK1 or SK4). While the IK1 and SK channels share roughly 40% homology, their expression patterns and pharmacology are widely disparate, consistent with their unique physiological functions (1). The human IK1 channel (hIK1) is widely expressed, being found in salivary gland, colon, bladder, stomach, lung, smooth muscle, red blood cells, T-cells, and placenta (2, 3) where it plays a crucial role in a variety of physiological functions. Indeed hIK1 plays a seminal role in modulating Ca2+-dependent transepithelial ion transport (4, 5), has recently been confirmed to be the Gardos channel of red blood cells (6), and has been proposed to play a role in both vascular remodeling (7) and in modulating vascular tone (8). Because of these critical physiological roles, the regulation of hIK1 has received a great deal of attention. For instance, hIK1 is directly gated by intracellular Ca2+ interacting with calmodulin that is constitutively associated with the COOH terminus of the channel (9-11). In addition, we have demonstrated that ATP activates hIK1 via cAMP-dependent protein kinase and that this activation relies upon a COOH-terminal domain overlapping the Ca2+-dependent, calmodulin-binding domain (12, 13). We have also demonstrated that hIK1 is inhibited by arachidonic acid (4), recently mapping the inhibitory arachidonic acid binding site to the pore of hIK1 (14). Furthermore the regulation of hIK1 by protein kinase C has been defined at the molecular level (15).

Given the potential clinical importance of this channel, it is not surprising that the pharmacology of hIK1 has similarly been extensively examined. We have demonstrated that IK and SK channels are activated by benzimidazolinones, including 1-ethyl-2-benzimdazolinone and DCEBIO, as well as the centrally acting muscle relaxants chlorzoxazone and zoxazolamine (5, 16). We (17) and others (2, 3) have demonstrated that clotrimazole is an inhibitor of IK channels. Recently Chandy and colleagues (18) mapped the molecular binding site for this blocker to the pore of hIK1.

In contrast to the studies on channel regulation and pharmacology, the assembly, trafficking, and cell surface expression of the hIK1 channel has been little studied. The hIK1 channel lacks a characteristic T1 assembly domain, conserved in many of the voltage-activated channels (19), and is therefore likely to possess a novel domain that aids in channel assembly and trafficking. Our laboratory recently defined a role for the COOH terminus of hIK1 in the trafficking of the channel to the cell membrane (20). We reported that a leucine zipper located in this region of the channel was crucial for channel folding and membrane trafficking but was not essential for channel assembly. Further Joiner et al. (21) demonstrated that the correct assembly of calmodulin with the COOH terminus of hIK1 was required for efficient delivery of the channel to the cell surface. To date, there have been no studies defining the role of the 26-amino acid cytoplasmic NH2 terminus in the assembly and trafficking of hIK1. Similar to the COOH terminus, the NH2 terminus of hIK1 contains a hydrophobic domain, including two potential leucine zippers, one of which extends into the first transmembrane domain, as well as a dileucine motif. As leucine zippers may form coiled-coil domains important in protein assembly (22) and dileucine motifs are known to be involved in protein trafficking (23) we determined the role of each of these motifs in the assembly, trafficking, and function of hIK1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified 5% CO2, 95% O2 incubator at 37 °C. Cells were transfected using LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were generated for all constructs by subjecting cells to antibiotic selection (1 mg/ml G418) 48 h post-transfection. Selection was typically complete within 14 days post-transfection. Following selection the concentration of G418 was reduced to 0.2 mg/ml. Note that clonal cell lines were not subsequently selected from this stable population of cells to avoid clonal variation.

Molecular Biology—The cDNA for hIK1 was originally provided by J. P. Adelman (Vollum Institute, Oregon Health Sciences University). HA (YPYDVPDYA) and Myc (EQKLISEEDL) epitope tags were inserted either into the second extracellular loop between Gly132 and Ala133 or at the COOH terminus, respectively, as described previously (20). All mutations in the HA-hIK1 channel were produced using the QuikChangeTM site-directed mutagenesis strategy (Stratagene). The fidelity of all constructs were confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (National Center for Biotechnology Information BLAST) with hIK1 (GenBankTM accession number AF022150 [GenBank] ).

Antibodies—For detection of HA-hIK1 by immunofluorescence (IF), immunoprecipitation (IP), and immunoblotting (IB) the following antibodies were obtained from the sources indicated (dilutions used are indicated): polyclonal HA (1:150) and monoclonal HA (1:1,000) (HA.11, Covance, Richmond, CA) and c-Myc (clone 9E10, 1:1,000, Roche Applied Science). The following secondary antibodies were obtained from the various sources indicated: biotin-conjugated goat anti-mouse IgG (1:200, Molecular Probes, Eugene, OR), streptavidin conjugated to Alexa Fluor® 488 (1:500, Molecular Probes), Cy3.18-conjugated goat anti-mouse IgG (1:3,000, Amersham Biosciences), and horseradish peroxidase-conjugated goat anti-mouse IgG (1:2,000, BD Biosciences).

Immunofluorescence—For IF labeling, HEK293 stable cell lines were grown on poly-L-lysine (Sigma)-coated glass coverslips for 24 h prior to labeling. Cell surface HA-hIK1 was labeled via sequential incubation in primary monoclonal HA antibody (1:1,000, 90 min) and secondary biotin-conjugated goat anti-mouse IgG (1:200, 90 min) followed by streptavidin conjugated to Alexa 488 (1:500; 90 min). All steps were performed at 4°C to prevent endocytosis of the channel. Following cell surface labeling, the cells were fixed with 2% paraformaldehyde, PBS and permeabilized with 0.1% Triton X-100, 2% paraformaldehyde, PBS, and intracellular localized HA-hIK1 was labeled sequentially with primary monoclonal HA antibody (1:1,000, 90 min), and secondary Cy3.18-conjugated goat anti-mouse IgG (1:3,000) antibody. Finally nuclei were labeled with Hoechst 33258 (Sigma). This approach allowed us to detect both cell surface and intracellular HA-hIK1 in the same cells as described previously (20). Cells were then subjected to laser confocal microscopy using a Leica TCSNT 3 laser 4 PMT system. To ensure maximal X-Y spatial resolution, sections were scanned at 1024 x 1024 pixels using sequential two-color image collection to minimize cross-talk between the channels imaged. All images shown in a single figure were scanned on the same day using identical settings. The images were then imported into Adobe Photoshop and combined into a single figure, and red-green-blue (RGB) brightness/contrast was adjusted identically for all panels.

Chemiluminescence—HEK cells were transiently transfected with the Myc-tagged and HA-tagged wild type hIK1 or L18A/L25A channel 24 h prior to the experiment. The cells were sequentially incubated in monoclonal HA antibody (1:1,000) for 90 min followed by goat anti-mouse horseradish peroxidase-conjugated IgG (1:2,000, BD Biosciences) for 1 h at 4 °C. The cells were then extensively washed, and the horseradish peroxidase-labeled proteins were detected using West Pico chemiluminescent substrate (Pierce) in conjunction with a TD20/20 luminometer (Turner, Sunnyvale, CA). Intensities are presented as a percentage of wild type.

Electrophysiology—During whole-cell patch clamp experiments the bath contained 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES (pH adjusted to 7.4 with NaOH). The pipette solution contained 130 mM KCl, 5 mM NaCl, 0.12 mM CaCl2, 4 mM MgCl2, 10 mM HEPES, and 0.2 mM EGTA (pH adjusted to 7.2 with KOH). All experiments were preformed at room temperature (22 °C) using stably transfected HEK293 cells plated the pervious day. Electrodes were fabricated from thin walled borosilicate glass (World Precision Instruments, Sarasota, FL), were pulled on a vertical puller (Narishige, Long Island, NY), and had a resistance of 1-4 megaohms. Currents were recorded using an Axon Instruments (Foster City, CA) 200B amplifier interfaced to a computer using a Digidata 1322A (Axon Instruments). Following establishment of the whole-cell configuration, voltage steps were applied using pClamp 8.2 software (Axon Instruments) from a holding potential of -60 mV at 250-ms pulses every 2 s from -100 to +80 mV in 20-mV increments to generate a current-voltage (I-V) relationship. Current was sampled at steady state (100 ms) for the purpose of evaluating current density. Current density (pA/pF) at 0 mV was calculated by dividing the current by the whole-cell capacitance.

Immunoprecipitation—For co-immunoprecipitation (Co-IP) of HA- and Myc-tagged hIK1 constructs, HEK293 cells were transiently transfected in 60-mm dishes using LipofectAMINE 2000 and 5 µg of each plasmid (total, 10 µg of DNA and 20 µl of lipid). When only a single construct was transfected (HA or Myc) empty pcDNA3.1(+) was included (5 µg) to maintain the same final concentration of plasmid and lipid in all dishes. Cells were lysed 18-24 h post-transfection with IP buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, Complete EDTA-free protease inhibitor (Roche Applied Science, pH 7.4), and protein concentrations were determined and normalized to achieve equivalent loading. Crude lysates were then precleared with protein A-Sepharose beads (Sigma) and incubated with polyclonal HA antibodies. Immune complexes were precipitated with protein A-Sepharose beads followed by sequential washes in IP buffer containing 500, 300, and 150 mM NaCl (2x) supplemented with 1x radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS). After the final wash, the pellet was resuspended in Laemmli sample buffer, and proteins were resolved by SDS-PAGE (12% gel) and transferred to nitrocellulose for immunoblot analysis as described below.

For cell surface IP (CS-IP), cells were grown to confluence in a 100-mm dish, and cell surface channels were labeled with polyclonal HA antibody (1:500) for 90 min at 4 °C followed by extensive washing to remove unbound antibody. As above, all steps were performed at 4 °C to prevent endocytosis of the channel and/or antibody. The cells were then lysed, protein concentrations were normalized, and the immune complexes were directly subjected to IP as detailed above. Following transfer to nitrocellulose, IB was performed using monoclonal HA antibody (1:1,000) as detailed below. In addition to the IP, 20 µg of protein was set aside following cell lysis for IB. In this way, we were able to confirm similar levels of protein expression in cells failing to correctly traffic HA-hIK1 to the cell surface.

Immunoblot Analysis—HEK293 cells were grown to confluence, lysed with IP buffer, separated by SDS-PAGE, and transferred to nitrocellulose. 20 µg of total protein was run on the gel unless otherwise noted in the text. Immunoblotting was then carried out as described previously (20). In experiments where band densities are plotted, the band intensities were determined using the UN-SCAN-IT gelTM automated digitizing system (Silk Scientific Corp., Orem, UT). Background intensity was determined for each immunoblot film used and subtracted from the intensity values. The percent changes in intensities were determined by dividing the experimental intensity by the wild type control intensity.

Chemicals—All chemicals were obtained from Sigma unless otherwise stated. DCEBIO was synthesized in the laboratory of R. J. Bridges (University of Pittsburgh) as described previously (24). Leupeptin, pepstatin, chloroquine, and lactacystin were purchased from Sigma and prepared as suggested by the manufacturer.

Statistics—All data are presented as means ± S.E. where n indicates the number of experiments. Statistical analysis was performed using a Student's unpaired t test. A value of p < 0.05 is considered statistically significant and is reported. The IF and other biochemical assays were repeated at least three times to ensure the fidelity of our results. The {tau} values were calculated using the exponential decay, single, two-parameter regression function of SigmaPlot 2001 (SPSS, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Role of the NH2 Terminus in the Expression, Trafficking, and Function of hIK1—We previously demonstrated that the correct trafficking of hIK1 to the cell surface was dependent upon a COOH-terminal leucine zipper (20). The 26-amino acid cytoplasmic NH2 terminus of hIK1 also contains two potential overlapping leucine zippers, resulting in the generation of a dileucine motif (see Fig. 1A). In our initial studies designed to elucidate the role of these motifs in the assembly and trafficking of hIK1 the entire cytoplasmic NH2 terminus was deleted from our HA epitope-tagged channel (HA-hIK1). We previously demonstrated that insertion of the HA epitope into the second extracellular domain of hIK1 allows us to monitor cell surface expression by IF and CS-IP while having no effect on the biophysical or regulatory properties of the channel (20). Following deletion of the cytoplasmic NH2 terminus, we evaluated cell surface expression by IF and CS-IP, total protein expression by IB, and channel function by the whole-cell patch clamp technique. Similar to previous results (20), we were able to detect expression of HA-hIK1 at the cell surface by IF localization (Fig. 1B, green labeling) as expected for the wild type channel. In addition, intracellular channel protein was clearly labeled (Fig. 1B, red labeling). However, following deletion of the cytoplasmic NH2 terminus (N-Del) there was a dramatic decrease in cell surface labeling of the channel, although intracellular channel protein was still labeled (Fig. 1C). This result suggests that the NH2 terminus of hIK1 is required for cell surface expression.



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  		FIG. 1.
NH2-terminal truncation inhibits cell surface expression of HA epitope-tagged hIK1. A, primary amino acid sequence of the NH2 terminus and first transmembrane domain of hIK1 (Met1-Ala44). Important structural motifs include the following: proximal leucine zipper (amino acids 5, 12, and 19 highlighted in bold), distal leucine zipper (amino acids 18, 25, 32, and 39 marked by the asterisks), and a dileucine motif (amino acids 18 and 19 indicated by the underline). B and C, confocal microscopy images of cells sequentially incubated with primary (HA), secondary (goat anti-mouse IgG-biotin), and tertiary (streptavidin-Alexa 488) antibodies at 4 °C to label plasma membrane HA-hIK1 (green). The cells were then fixed in 2% paraformaldehyde, PBS, and permeabilized with 0.1% Triton X-100, PBS to label intracellular channel protein (red). B, HA-hIK1; C, HA-tagged NH2-terminal deletion construct (N-Del). D, CS-IP and IB. Top, CS-IP for HA-hIK1 (lane 1) and N-Del (lane 2) shows that the N-del construct is not expressed at the cell surface. Bottom, immunoblot with 20 µg of total protein loaded in each lane and blotted with monoclonal HA antibody demonstrates similar levels of expression of both constructs. The immunoblot and CS-IP blot are representative of three experiments. E, DCEBIO-stimulated current density (pA/pF) plotted for HA-hIK1 and N-Del at 0 mV (mean ± S.E.; number of experiments is indicated in parentheses). There is a significant decrease in current density in cells expressing the N-Del construct compared with wild type (*, p < 0.05). TM, transmembrane domain.

 
To quantitatively evaluate both cell surface and total protein expression of hIK1 following deletion of the NH2 terminus we performed CS-IP in conjunction with IB. Consistent with our IF data, we demonstrated by CS-IP that HA-hIK1 was abundantly expressed at the cell surface (Fig. 1D, lane 1), while we were unable to detect surface expression of the N-Del channel (Fig. 1D, lane 2). The immunoblot analysis confirmed that HA-hIK1 and N-Del were expressed at similar levels consistent with the IF data (Fig. 1D), indicating that the lack of cell surface expression is not due to a lack of protein expression. Finally we confirmed a lack of functional N-Del channel expression using the whole-cell patch clamp technique. As shown in Fig. 1E, HA-hIK1 could be activated by the pharmacological channel opener DCEBIO as described previously (16, 24). In eight experiments, DCEBIO (10 µM) increased current density an average of 107 ± 22 pA/pF in HA-hIK1-expressing cells. Consistent with our IF and CS-IP results, cells expressing the N-Del channel failed to respond to DCEBIO (0.6 ± 0.3 pA/pF, n = 15), indicative of no functional channels being expressed at the cell surface. These results clearly demonstrate a role for the NH2 terminus of hIK1 in the functional expression of this channel at the plasma membrane.

The Leu5/Leu12/Leu19 Leucine Zipper Does Not Contribute to hIK1 Assembly, Trafficking, or Function—As shown in Fig. 1A, there are multiple leucine-based motifs in the cytoplasmic NH2 terminus of hIK1 that may contribute to the lack of cell surface expression observed following deletion. One such motif is a potential leucine zipper at amino acid positions Leu5/Leu12/Leu19 (in Fig. 1A these leucines are shown in boldface type). We therefore mutated the first two leucines in this potential zipper motif to alanines (L5A/L12A) and determined the effect of this mutation on trafficking and function. As assessed by CS-IP, IB, and whole-cell current density measurements the L5A/L12A mutation had no significant effect on hIK1 expression or function (data not shown). These results indicate that this proximal NH2-teminal leucine zipper is not required for correct expression and function of hIK1.

Role of the NH2-terminal Leucine Zipper (Leu18/Leu25/Leu32/Leu39) and Dileucine (Leu18/Leu19) Motifs in hIK1 Expression and Function—The NH2 terminus of hIK1 contains a second potential leucine heptad repeat that extends into the first transmembrane domain (Leu18/Leu25/Leu32/Leu39). In Fig. 1A, the leucines comprising this heptad repeat are highlighted by asterisks. Interestingly a dileucine motif is formed from the last leucine in the first heptad repeat and the first leucine in this second heptad repeat, i.e. Leu18/Leu19 (Fig. 1A, underline). To evaluate the role of this second potential leucine zipper as well as the dileucine motif, we constructed a series of single and double alanine substitution mutations and determined their effect on cell surface expression, total protein expression, and function. As shown in Fig. 2, mutation of Leu18 to Ala (L18A) had no significant effect on surface expression as assessed by both IF (Fig. 2A) and CS-IP (Fig. 2B) or function as assessed by whole-cell patch clamp current density measurements (Fig. 2C; HA-hIK1, 157 ± 18, n = 50; L18A, 137 ± 38 pA/pF, n = 10; p = 0.65). In contrast, the additional individual alanine substitutions L19A and L25A resulted in clear reductions in channel surface expression (Fig. 2, A and B), total protein expression (Fig. 2B), and functional channel expression (Fig. 2C; L19A, 49 ± 23 pA/pF, n = 10; L25A, 68 ± 15 pA/pF, n = 6; p < 0.05). These results suggest that the NH2-terminal leucines in hIK1 are crucial for normal channel expression and function.



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  		FIG. 2.
Mutations in the NH2-terminal dileucine and leucine zipper motifs of hIK1 affect expression and trafficking. A, confocal microscopy images are shown for wild type HA-hIK1, L18A, L19A, and L18A/L19A in the top panel and for L25A, L18A/L25A, L25A/L32A, and L32A/L39A in the bottom panel. Cells were sequentially incubated with primary (HA), secondary (goat anti-mouse IgG-biotin), and tertiary (streptavidin-Alexa 488) antibodies at 4 °C to label plasma membrane HA-hIK1 (green label). The cells were then fixed in 2% paraformaldehyde, PBS and permeabilized with 0.1% Triton X-100, PBS to label intracellular channel protein (red label). Note that mutation of the dileucine (L18A/L19A) and leucine zipper (L18A/L25A) motifs resulted in a dramatic reduction in both cell surface and intracellular labeling. B, IB and CS-IP. Top, immunoblot with 20 µg of protein for each construct loaded and blotted with monoclonal HA antibody. The L18A/L19A and L18A/L25A mutated channels resulted in a near complete loss of protein expression. Bottom, CS-IP confirms the complete loss of cell surface expression of the L18A/L19A and L18A/L25A mutated channels as well as dramatic reductions in cell surface expression for additional mutations in the leucine zipper (L25A, L25A/L32A, and L32A/L39A) and dileucine (L18A/L19A) motifs. Blots are representative of six separate experiments each. C, whole-cell DCEBIO-stimulated current densities (pA/pF) for each construct at 0 mV (mean ± S.E., number of experiments is indicated in parentheses). Current densities for leucine mutated channels that were significantly different from wild type channel are indicated (*, p < 0.05; **, p < 0.01; and ***, p < 0.001).

 
We next constructed double mutations in both of these motifs and determined their effects on channel expression. Similar results to those above were obtained when the double alanine mutations L25A/L32A and L32A/L39A were introduced into the NH2-terminal leucine zipper of HA-hIK1. That is, there was a clear decrease in both cell surface channel (Fig. 2, A and B) and total protein expression (Fig. 2B). Whole-cell patch clamp experiments (Fig. 2C) confirmed a significant reduction in current density for these double mutations (L25A/L32A, 75 ± 32 pA/pF, n = 8; L32A/L39A, 74 ± 24 pA/pF, n = 10; p < 0.05) when compared with wild type HA-hIK1. In contrast to these rather modest effects, introducing the double mutation L18A/L25A or L18A/L19A into the leucine zipper or the dileucine motif, respectively, resulted in a complete loss of cell surface expression (Fig. 2, A and B). Indeed we were unable to detect any IF signal following mutation of L18A/L25A either at the cell surface or inside the cell (note the complete absence of red IF signal), whereas a very weak signal, corresponding to intracellular protein, was detected following mutation of L18A/L19A (Fig. 2A). The reason for the total loss of intracellular IF signal is apparent when one considers the immunoblot shown in Fig. 2B. That is, mutation of L18A/L19A produced a near complete loss of total protein, and no protein was detected following mutation of L18A/L25A. These biochemical studies were confirmed electrophysiologically by the absence of a DCEBIO-induced current response in both L18A/L19A (11 ± 4 pA/pF, n = 13; p < 0.01) and L18A/L25A (0.3 ± 0.2 pA/pF, n = 8; p < 0.001; Fig. 2C). These results demonstrate that both the NH2-terminal dileucine motif (Leu18/Leu19) and the leucine zipper (Leu18/Leu25/Leu32/Leu39) are critical for maintaining hIK1 protein expression.

Rate of Channel Degradation—Our results above demonstrate a dramatic decrease in steady-state protein levels for the L18A/L19A and L18A/L25A mutations compared with wild type HA-hIK1, indicative of an enhanced degradation of these mutated channels. Based on these results, we determined the protein half-life of HA-hIK1, L18A/L19A, and L18A/L25A channels in the presence of cycloheximide (400 µg/ml), an inhibitor of protein synthesis. As shown in Fig. 3A, ~70% of wild type HA-hIK1 channel was degraded with first-order kinetics, having a time constant, {tau},of3.2 ± 0.8h(inset, dashed line). The remainder of the HA-hIK1 protein remained stable for greater than 24 h for reasons that are not apparent. Based on our steady-state results (Fig. 2B), we expected to observe a more rapid rate of channel degradation following mutation of L18A/L19A and L18A/L25A. This was indeed the case with the L18A/L19A channel being degraded with a {tau} of 64.7 ± 13.3 min (Fig. 3B) and the L18A/L25A channel being more rapidly degraded with a {tau} of only 19.0 ± 1.3 min (Fig. 3C). These results clearly demonstrate that full-length L18A/L19A and L18A/L25A channels were being synthesized, but their rate of degradation was enhanced 3- and 10-fold, respectively, compared with HA-hIK1.



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  		FIG. 3.
Decay of wild type, L18A/L19A, and L18A/L25A hIK1 channel total protein expression. Protein degradation was measured following treatment with cycloheximide (400 µg/ml) for various periods of time as indicated. Protein samples were loaded (20 µg of HA-hIK1, 80 µg of L18A/L19A, and 100 µg of L18A/L25A) and separated by SDS-PAGE. The resulting immunoblot films were digitized, and band intensities for the varying time points were determined as a percent change from time 0. The decrease in total protein was fit to an exponential decay function, and the time constant was determined ({tau} ± S.E., n = 4 for each construct). A, HA-hIK1 total protein decay over a 24-h time period. Inset, exponential fit to first 6 h of decay. B, L18A/L19A degradation. C, L18A/L25A degradation.

 
Mutation of the NH2-terminal Leucine Zipper and Dileucine Motif Results in Proteasomal Degradation—Based on the above results, we determined the cellular compartment where L18A/L19A and L18A/L25A are subject to degradation. For these studies we utilized lactacystin, an inhibitor of proteasomal degradation, as well as a combination of leupeptin and pepstatin, which together inhibit lysosomal degradation of proteins by inhibiting serine/cysteine proteases as well as cathepsins B and D (25). As shown in Fig. 4B, neither proteasomal nor lysosomal inhibitors affected steady-state HA-hIK1 channel expression. Similarly leupeptin and pepstatin had little effect on protein expression of either L18A/L19A or L18A/L25A (Fig. 4, A, C, and D), suggesting these channels are not primarily subjected to lysosomal degradation. In contrast, inhibition of the proteasomal degradation pathway with lactacystin resulted in a dramatic increase in steady-state protein levels for both L18A/L19A and L18A/L25A to levels similar to wild type HA-hIK1 protein (Fig. 4, A, C, and D). These results unequivocally demonstrate that mutation of these NH2-terminal leucines results in a channel that is targeted for rapid proteasomal degradation. We further evaluated whether this increased protein expression would result in an enhanced level of cell surface expression. As shown in Fig. 4A, despite the large increases in L18A/L19A and L18A/L25A protein expression in the presence of lactacystin (lower panel), we failed to detect any protein at the cell surface (upper panel). These results indicate that mutations in the NH2-terminal leucines of hIK1 affect protein expression as well as channel assembly and/or trafficking as full-length channel is not expressed at the cell surface.



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  		FIG. 4.
Mutation of the dileucine (L18A/L19A) and leucine zipper (L18A/L25A) motifs results in proteasomal degradation. CS-IP and IB for wild type HA-hIK1, L18A/L19A, and L18A/L25A either untreated or treated with lactacystin (Lac) (100 µM), chloroquine (Chl) (100 µM), or leupeptin (15 µM)/pepstatin (15 µM) (L/P) for 16 h. CS-IP shows that inhibition of the proteasome with lactacystin does not increase cell surface expression of the L18A/L19A or L18A/L25A constructs (lanes 3 and 7), although total protein expression (IB) was dramatically increased (lanes 3 and 7). Chloroquine, an inhibitor of endocytosis, increased both total protein expression (IB) (20 µg of protein loaded) and cell surface expression (CS-IP) of the L18A/L19A mutation (lane 4) while having little effect on L18A/L25A (lane 8). Finally inhibition of lysosomal degradation with leupeptin/pepstatin had no effect on L18A/L25A (lane 9) and had only a modest effect on L18A/L19A expression (lane 5). B, C, and D, densitometry quantification of protein expression levels following 16-h inhibitor treatment are given for HA-hIK1 (n = 7) (B), L18A/L19A (n = 6) (C), and L18A/L25A (n = 5) (D) channels. Plots are presented as percent change from non-treated cells.

 
Chloroquine Rescues Cell Surface Expression of L18A/L19A—Chloroquine has been shown to have a wide array of effects on protein degradation and trafficking, including the inhibition of endocytosis (26, 27). Storey et al. (28) demonstrated that chloroquine blocked the endocytic retrieval of voltage dependent Na+ channels in mammalian neurons. Therefore, we determined whether chloroquine would increase the cell surface expression of L18A/L19A and L18A/L25A channels. As shown in Fig. 4A, incubating cells expressing the L18A/L25A channel in chloroquine had only a modest effect on protein expression and no effect on cell surface expression. In contrast, chloroquine prevented the degradation of L18A/L19A, and this channel was abundantly expressed at the cell surface. We interpret these results to indicate that small amounts of L18A/L19A are normally escaping the degradation pathway and correctly trafficking to the plasma membrane; inhibition of endocytosis by chloroquine allows these channels to be detected.

Mutation of L18A/L19A Does Not Alter Cell Surface Residence Time—Our results demonstrate that L18A/L19A has the ability to traffic to the cell surface in the presence of chloroquine. We previously demonstrated that mutations in the COOH-terminal leucine zipper resulted in a channel that failed to correctly traffic to the plasma membrane and that this trafficking defect could be corrected by growing the cells at the permissive temperature of 27 °C (20). Thus, we determined whether growing cells at 27 °C would allow for cell surface expression of L18A/L19A. Similar to our results with chloroquine, following incubation at 27 °C for 16 h L18A/L19A was detected at the plasma membrane by CS-IP (data not shown). These results suggest that mutation of the NH2-terminal dileucine motif in hIK1 results in a subtle folding defect that can be overcome by growing the cells at a reduced temperature, altering trafficking of the channel to the cell surface.

As dileucine motifs are known to be involved in the endocytic retrieval of proteins, we determined whether mutation of the dileucine in hIK1 would alter residence time of the channel at the cell surface. For these studies, we incubated the cells at 27 °C for 16 h to promote expression of the L18A/L19A mutation at the cell surface. The cell surface channel was then labeled with antibody at 4 °C after which the cells were incubated at 37 °C for various periods of time. Subsequently the channel was immunoprecipitated and quantified by densitometry. Our results demonstrated no change in cell surface retrieval between wild type and L18A/L19A channels, having {tau} values of 3.8 ± 0.7 and 3.7 ± 0.7 h, respectively. These results demonstrate that once L18A/L19A has been correctly trafficked to the plasma membrane it exhibits internalization characteristics similar to wild type HA-hIK1.

The L18A/L25A Mutation Affects Assembly of hIK1—The rapid targeting of the L18A/L25A channels for degradation may be the result of the inability of the channel to correctly assemble into a tetrameric complex. To address the question of channel assembly, a Myc epitope-tagged hIK1 was constructed. Combinations of HA-hIK1 and Myc-hIK1 were transiently co-transfected into HEK cells in the presence and absence of lactacystin, and these were subsequently subjected to Co-IP. A stable HA-hIK1·Myc-hIK1 heterotetramer would contain both channel subunits when precipitated with the HA antibody and blotted with Myc antibody to detect the channel. As shown in Fig. 5, we were able to co-immunoprecipitate HA-hIK1 and Myc-hIK1 following co-transient transfection of these two channels (lane 3). As shown in lane 4, transient transfection of HA-tagged L18A/L25A alone resulted in no Co-IP due to the lack of Myc-tagged channel (top panel). As in Fig. 2, very little protein was detected in the immunoblot with the HA antibody (bottom panel), demonstrating that further overexpression within this transient transfection system cannot overwhelm the degradative destiny of this mutated channel. Co-transfection of HA-tagged L18A/L25A with wild type Myc-hIK1, in the presence of lactacystin, revealed that this mutated channel is capable of assembling with wild type channel (lane 5). However, the ability to co-assemble was clearly compromised despite the high level of HA-tagged L18A/L25A protein present in these cells (lane 5, IB). Further, if HA-tagged L18A/L25A was co-transfected with Myc-tagged L18A/L25A, virtually no product was detected following Co-IP (lane 6). Again lactacystin was used to ensure high levels of expression of these mutated channels (lane 6, IB). These results clearly demonstrate that leucines in the cytoplasmic NH2 terminus of hIK1 are required for efficient tetramer formation.



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  		FIG. 5.
Multimer formation of wild type and L18A/L25A channels. Top, HA- and Myc-tagged hIK1 were transiently transfected, either alone or in combination (lanes 1-3), into HEK293 cells and subjected to Co-IP. Immunoprecipitations were done using polyclonal HA antibody with monoclonal HA antibody being used for the subsequent IB. For IB alone 20 µg of protein was loaded in each lane. Lanes 1 and 2 are controls for antibody specificity. As shown in lane 3, Myc-hIK1 was co-precipitated with HA-hIK1. Transfection of HA-L18A/L25A alone resulted in no Co-IP (lane 4) as well as a reduced level of protein expression as expected (lane 4, IB). Transfection of a wild type Myc-hIK1 with HA-L18A/L25A channel, in the presence of lactacystin to increase total L18A/L25A protein expression (lane 5, IB), demonstrates co-assembly of the wild type and mutated channel (lane 5, Co-IP). Note, however, that this is dramatically reduced relative to the co-assembly observed with two wild type channels (lane 3). Co-transfection of both L18A/L25A mutated HA- and Myc-tagged channels, in the presence of lactacystin to increase total L18A/L25A protein expression (lane 6, IB), resulted in a complete loss of channel assembly as assessed by Co-IP (lane 6). Blots are representative of three separate experiments. mono, monoclonal; poly, polyclonal; Lac, lactacystin.

 
Our results above indicate that the L18A/L25A mutation is still capable of assembling with wild type channel, although this is clearly compromised. Thus, we determined whether this co-assembly with L18A/L25A would inhibit cell surface expression of wild type channel. For these studies we transiently co-transfected wild type and mutated channel into HEK cells and evaluated cell surface expression of the HA-tagged channel using both IF as well as a chemiluminescence assay (29). As shown in Fig. 6A, co-transfecting wild type HA- and Myc-tagged hIK1 channels resulted in a clear IF signal corresponding to cell surface HA-hIK1 (left panel, green). Co-transfection of Myc-tagged L18A/L25A with wild type HA-hIK1 attenuated the cell surface, HA-associated, IF signal (middle panel). These results are consistent with the mutated channel co-assembling with wild type channel, precluding the normal trafficking of the channel to the cell surface. Finally we determined whether co-transfecting wild type Myc-hIK1 would rescue the expression of HA-tagged L18A/L25A, resulting in its expression at the cell surface. Fig. 6A shows that this is not the case; the HA-tagged L18A/L25A construct was unable to traffic to the membrane when co-assembled with a wild type Myc-tagged channel (right panel, no green signal). As shown by the immunoblot in Fig. 6B, co-transfecting wild type Myc-hIK1 resulted in an increased expression of HA-tagged L18A/L25A (lane 3) when compared with the HA-tagged L18A/L25A alone (lane 2). We interpret this to indicate that co-assembly of these channels stabilizes the mutated channel such that its degradation is slowed. Despite the increased expression of HA-tagged L18A/L25A this channel failed to traffic to the cell surface (Fig. 6A, right panel). These effects were more quantitatively addressed using the chemiluminescence-based assay devised by Jan and colleagues (29). As shown in Fig. 6C, co-transfection of Myc-tagged L18A/L25A with HA-hIK1 resulted in a significant decrease in cell surface expression of HA-hIK1. This result could be interpreted to suggest that this leucine zipper mutation does not act as a dominant negative for channel expression as the cell surface signal was reduced only 54% rather than the 94% expected for a true dominant negative effect of a tetrameric complex. However, this result is explained if one considers the fact that, despite equivalent amounts of DNA being transfected, the L18A/L25A channel will be more rapidly degraded. Thus, one would expect a greater number of homotetrameric HA-hIK1 channels as these subunits would be expressed in greater abundance. Similar to our IF data, wild type Myc-hIK1 was not capable of increasing HA-tagged L18A/L25A expression at the cell surface. In total, these results indicate that mutation of the NH2-terminal leucine zipper does not preclude its insertion into the channel complex. However, incorporation of a mutated subunit dramatically compromised the ability of the channel to correctly traffic to the cell surface.



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  		FIG. 6.
Interaction of wild type and L18A/L25A hIK1 channels alters cell surface and total protein expression. A, confocal microscopy images of cells sequentially incubated with primary (HA), secondary (goat anti-mouse IgG-biotin), and tertiary (streptavidin-Alexa 488) antibodies at 4 °C to label plasma membrane HA-hIK1 (green label). Left, co-transfection of wild type HA-hIK1 and Myc-hIK1 resulted in clear cell surface expression of HA-hIK1 (green label). Middle, co-transfection of Myc-L18A/L25A with HA-hIK1 inhibited expression of HA-hIK1 at the cell surface. Right, co-transfection of HA-L18A/L25A and Myc-hIK1 channels resulted in no cell surface labeling of the HA epitope. For these cells the nuclei are labeled with Hoescht 33258 (blue). B, immunoblot (20 µg of protein) showing protection of L18A/L25A degradation when co-transfected with wild type Myc-tagged channel (lane 3). C, cell surface expression of HA-hIK1 was quantified by luminometry following transfection with the constructs indicated (n = 3 separate sets of data points). Similar to the IF data in A, L18A/L25A-Myc-hIK1 inhibited expression of wild type HA-hIK1 at the cell surface. Data are presented as percent change from wild type HA-hIK1 + Myc-hIK1 co-transfection.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Assembly of K+ channels into homo- or heterotetramers requires the interaction of assembly or tetramerization domains. These domains are typically conserved within a gene family, thereby allowing heterotetramerization to occur, whereas the formation of functional channels across gene families is excluded. The domains required for assembly of the intermediate (IK) and small (SK) conductance Ca2+-activated K+ channels have been little studied. However, two recent reports have demonstrated a role for the COOH terminus in this process. That is, Joiner et al. (21) recently demonstrated that calmodulin, a known regulator of the hIK1 channel, is required for assembly and trafficking. Also, we recently demonstrated that the distal COOH terminus of hIK1 is capable of self-assembly and that this is dependent upon an intact leucine zipper motif (20). To date, the role of the NH2 terminus on the trafficking and function of hIK1 has not been explored. However, the cytoplasmic NH2 termini of K+ channels are well known to be involved in channel assembly, trafficking, and regulation (30-32).

In this study, we demonstrate that deletion of the cytoplasmic NH2 terminus of hIK1 results in an inability of the truncated channel to traffic properly to the cell surface (Fig. 1). Although the cytoplasmic NH2 terminus of hIK1 is but 26 amino acids in length it contains numerous motifs that may be crucial for channel assembly, trafficking, or function including two potential leucine zippers, a dileucine motif, and a highly charged cluster of amino acids (Fig. 1A). We demonstrate that both the dileucine motif (Leu18/Leu19) as well as the leucine zipper motif (Leu18/Leu25/Leu32/Leu39) are required to prevent rapid channel degradation via the proteasome and that the leucine zipper is required for efficient channel tetramerization.

Previous studies have not addressed the question of hIK1 degradation rates or pathways or the residence time of the channel at the cell surface. When new protein synthesis was halted using cycloheximide there was an initial exponential decline in channel protein expression levels ({tau} = 3.2 ± 0.8 h); this decay was then followed by a stabilization of protein levels at 30% of the initial amount (Fig. 3A). Why this remaining protein is not subject to degradation following cycloheximide treatment is unclear. We were also unable to define the intracellular compartment responsible for degradation of wild type hIK1 channels. That is, inhibitors of either proteasomal (lactacystin) or lysosomal (leupeptin/pepstatin) degradation had no affect on steady-state hIK1 protein levels (Fig. 4B). In contrast to the wild type channel, mutation of either the NH2-terminal dileucine or leucine zipper motifs resulted in rapid lactacystin-dependent proteasomal degradation (Fig. 4). In addition, we determined the rate of hIK1 internalization from the plasma membrane as this would be crucial in defining the physiological role of the channel. We observed that hIK1 was removed from the cell surface with a {tau} of 3.8 h. It is interesting to note that the time course for channel retrieval from the cell surface is similar to the time course for total protein degradation. Thus, the degradation rate observed in the presence of cycloheximide may represent the loss of cell surface protein.

Role of the NH2-terminal Dileucine Motif—Mutation of the dileucine motif at amino acids Leu18 and Leu19 results in a channel that is degraded 3-fold faster than wild type channel (Fig. 3B). Further studies revealed that this channel is targeted for proteasomal degradation as lactacystin markedly increased protein expression (Fig. 4A). While we did not explicitly evaluate channel assembly for this construct, two results argue against a direct effect on channel tetramerization. First, addition of chloroquine, an inhibitor of receptor-mediated endocytosis (26, 27), resulted in abundant expression of the channel at the cell surface (Fig. 4A). The most parsimonious interpretation of this result is that L18A/L19A hIK1 is capable of trafficking to the cell surface, albeit at a level dramatically reduced compared with wild type channel, and that this channel accumulates following inhibition of endocytosis. An identical result has previously been reported for the voltage-gated Na+ channel (28). Chloroquine, however, has also been demonstrated to cause the inhibition of lysosomal function (33-35). The result observed with the L18A/L19A cell surface expression is unlikely to be caused by an inhibition of lysosomal degradation as cell surface expression of L18A/L19A hIK1 was unaffected by the additional lysosomal inhibitors leupeptin and pepstatin (Fig. 4). A second result arguing against a role for this dileucine motif being involved in channel tetramerization is the observation that reducing the temperature at which the cells were grown to 27 °C caused channels to be correctly localized to the cell surface (data not shown). In both of these experiments channels must have been correctly assembled into a tetrameric complex to be expressed at the cell surface. Thus, we interpret our results to indicate that mutation of the NH2-terminal dileucine motif results in a channel that correctly assembles but fails to efficiently traffic to the cell surface and is targeted for proteasomal degradation under normal growth conditions.

Dileucine motifs are known to be involved in protein recycling between the plasma membrane and endocytic vesicles as well as sorting at the trans-Golgi network (23, 36-38). Our results argue against a role for this dileucine motif in endocytic retrieval from the cell surface. That is, subsequent to increasing cell surface expression of L18A/L19A hIK1 via incubation at a permissive temperature (27 °C) this channel was internalized at a rate not different from wild type hIK1 ({tau} = 3.8 and 3.7 h, respectively). Indeed if the dileucine acted as an endocytic retrieval signal it would be expected that the L18A/L19A channel would remain at the cell surface for a longer duration of time. Our data do not allow us to determine whether the forward transport rates out of the endoplasmic reticulum or Golgi have been affected by this mutation. The inability of the channel to traffic may be the result of the dileucine motif being unable to interact with an adaptor protein, or it may simply be caused by an alteration in protein folding that results in the channel being targeted for degradation.

Role of the NH2-terminal Leucine Zipper Motif—In addition to a dileucine motif, the NH2 terminus of hIK1 also contains two potential leucine zippers. As leucine zippers are known to be involved in protein-protein interactions (39), these zipper motifs may play a role in the assembly of hIK1. Our data suggests that a leucine zipper in the cytoplasmic NH2 terminus is critical for the efficient assembly of hIK1 into a tetrameric complex. Co-immunoprecipitation showed that co-transfection of an HA epitope- and Myc epitope-tagged form of the wild type channel resulted in the formation of a channel complex in which both epitope tags could be detected (Fig. 5). However, transfection with an HA- and Myc-tagged L18A/L25A channel resulted in a loss of channels expressing both epitope tags. This result clearly defines the role of these NH2-terminal amino acids in the assembly of hIK1 into a multimeric complex. Importantly these results were observed under conditions where protein expression levels were elevated by inhibiting protein degradation with lactacystin (Fig. 5). However, additional data suggest that these amino acids are not solely responsible for channel assembly. First, our Co-IP studies demonstrate that wild type and mutated channels are capable of co-assembly (Fig. 5). Second, expression of a mutated channel (L18A/L25A) with a wild type channel results in the suppression of surface-expressed, non-mutated channel (Fig. 6). For this result to occur the mutated channel had to interact with the wild type channel. Finally we demonstrate that expressing wild type channel with mutated channel actually increases protein expression of the mutated channel (Fig. 6). This similarly suggests that the two channel subunits interacted such that the mutated subunit is stabilized, resulting in a reduced degradation rate.

It is interesting to note that although the leucine at amino acid position 18 is involved in both sets of mutations being studied we believe that the dileucine and leucine zipper motifs are indeed distinct but overlapping. This is based on the observation that the L18A/L19A channel, while being predominantly targeted for degradation, can be correctly trafficked to the plasma membrane in the presence of chloroquine (Fig. 4) or when the cells are grown at a permissive temperature (data not shown). In contrast, we were unable to demonstrate expression of L18A/L25A at the cell surface under any conditions. Finally L18A/L19A hIK1 channels are capable of assembly as evidenced by their cell surface expression, whereas L18A/L25A hIK1 channels failed to correctly assemble into multimeric complexes (Fig. 5). In conclusion, we demonstrate that the cytoplasmic NH2 terminus of hIK1 contains motifs required for channel assembly. These data represent the first demonstration of a role for the cytoplasmic NH2 terminus in the assembly, trafficking, or regulation of the IK/SK family of K+ channels.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DK54941 (to D. C. D.), the University of Otago Dean's Fund (to K. L. H.), and the University of Otago, Department of Physiology, for sabbatical support (to K. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S312 BST, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8755; Fax: 412-648-8330; E-mail: dd2{at}pitt.edu.

1 The abbreviations used are: SK, small conductance Ca2+-activated K+ channel; IK, intermediate conductance Ca2+-activated K+ channel; hIK1, human IK1 channel; IP, immunoprecipitation; Co-IP, co-immunoprecipitation; CS-IP, cell surface immunoprecipitation; HEK, human embryonic kidney; HA, hemagglutinin; IF, immunofluorescence; IB, immunoblotting; PBS, phosphate-buffered saline; pF, picofarad; DCEBIO, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; N-Del, deletion of the cytoplasmic NH2 terminus; {tau}, time constant. Back



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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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