Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper.

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of non-functional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu(406) (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27 degrees C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

The second messenger-dependent regulation of hIK1 has been extensively investigated at the molecular level during the past several years. That is to say both IK and SK channels are known to constitutively bind calmodulin and both have Ca 2ϩindependent and Ca 2ϩ -dependent calmodulin binding domains within the first 100 amino acids of the C-terminal tail (13,16,17). Indeed, it is the binding of Ca 2ϩ to EF hands 1 and 2 of calmodulin that results in the Ca 2ϩ -dependent gating of these channels (17). More recently, the crystal structure of calmodulin bound to the proximal C terminus of rSK2 was determined, demonstrating that calmodulin cross-links two C-terminal tails in the presence of Ca 2ϩ (18). We demonstrated recently (19) that the IK, but not the SK, channel was activated by ATP-dependent phosphorylation and that this was independent of consensus kinase phosphorylation sites on hIK1. We further mapped the domain for this kinase-dependent regulation to a 14-amino acid region (Arg 355 -Met 368 ) overlapping the Ca 2ϩ -dependent calmodulin-binding domain, suggesting this may be the site of additional protein-protein interactions (20). Similarly, PKC has been shown to acutely regulate IK1, and this is independent of consensus PKC phosphorylation sites (21).
In contrast to these results on the second messenger regulation of IK1, the molecular motifs required for tetramerization of hIK1 in the endoplasmic reticulum and the sorting motifs required for correct plasma membrane localization have been little studied. We demonstrated previously that truncation of the 427-amino acid hIK1 at Lys 402 resulted in the complete loss of channel function, as assessed by patch clamp techniques (20). This loss of function could result from either a failure of hIK1 to traffic to the plasma membrane or the expression of non-functional channels. To delineate among these possibilities an HA epitope was inserted into the extracellular loop between transmembrane domains S3 and S4 such that cell surface expression could be monitored by immunofluorescence (IF) and cell surface immunoprecipitation (CS-IP) techniques, whereas the function of hIK1 could be measured using the whole-cell patch clamp technique. We demonstrate that a C-terminal leucine zipper, distal to the calmodulin-binding domain, is required for the trafficking of hIK1 to the plasma membrane and that mutations of the leucine zipper alter the assembly of the distal C-terminal tail of hIK1. In contrast, this conserved leucine zipper is not required for the correct trafficking of another gene family member, SK3, to the plasma membrane.
Addition of either the Xpress (DLYDDDDK) or V5 (GKPIPNPLL-GLDST) epitope tag to the last 59 amino acids of the C terminus of hIK1 (Val 369 -Lys 427 ; C59) were generated by PCR amplification of the C59 fragment and subcloning into either pcDNA4/HisMax for Xpress or pcDNA3.1-V5/His-TOPO for V5 (Invitrogen). In total, 45 amino acids were added during the generation of the V5-C59 construct, whereas 35 amino acids were added in generating the XP-C59 construct, thereby allowing separation via SDS-PAGE.
The fidelity of all constructs utilized in this study was confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (NCBI BLAST) with hIK1 (GenBank TM accession number AF022150) or rSK3 (GenBank TM accession number U69884).
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% CO 2 , 95% O 2 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 in order to avoid clonal variation.
Electrophysiology-During whole-cell patch clamp recording, the bath contained (in mM) 140 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , and 10 HEPES (pH adjusted to 7.4 with NaOH). The pipette solution contained (in mM) 144 KCl, 7.6 CaCl 2 , 5.6 MgCl 2 , 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH) to achieve an intracellular free Ca 2ϩ concentration of 300 nM. For whole-cell recording of SK channels, the bath contained (in mM) 164.5 KCl, 2 CaCl 2 , 1 MgCl 2 , and 5 HEPES (pH adjusted to 7.4 with KOH). The pipette solution contained (in mM) 135 KCl, 8.7 CaCl 2 , 2 MgCl 2 , 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with KOH) to achieve an intracellular free Ca 2ϩ concentration of 1 M. All experiments were performed at room temperature. Currents were recorded using a List EPC-7 amplifier (Medical Systems, Greenvale, NY). Electrodes were fabricated from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL), pulled on a vertical puller (Narishige, Long Island, NY) and had a resistance of 1-4 megohms. Following establishment of the whole-cell configuration, voltage steps were applied from a holding potential of Ϫ60 mV at 250-ms pulses every 2 s from Ϫ100 to ϩ80 mV in 20-mV increments to generate a currentvoltage (I-V) relationship. Current was sampled at steady state (50 ms) for the purpose of evaluating current density. Similar I-Vs were generated following stimulation with DCEBIO (15) (10 M) and inhibition with clotrimazole (CLT; 3 M) or apamin (30 nM) for hIK1 or rSK3 channels, respectively. The pharmacological opener, DCEBIO, was utilized to eliminate any variability that could be caused by artificially raising Ca 2ϩ in the presence of pipette EGTA. Current density (pA/pF) was calculated by dividing the difference between DCEBIO-stimulated and CLT-or apamin-blocked current (pA) by the cell capacitance (pF) at a voltage of Ϫ20 or ϩ40 mV for hIK1 and rSK3 channels, respectively. Data analysis was performed using pCLAMP (version 5.5, Axon Instruments, Foster City, CA). For all HA-hIK1 constructs, whole-cell current densities were obtained on at least 10 cells to account for any variation in expression across the stable cell lines. For IF labeling, HEK293 stable cell lines were grown on poly-L-lysine (Sigma)-coated glass coverslips for 24 h prior to labeling. For detection of cell surface HA-hIK1, the cells were washed in ice-cold PBS, blocked in 1% BSA (3 ϫ 5 min) followed by goat serum (10% for 20 min). HA-hIK1 was then labeled sequentially by incubating in 1°(primary) monoclonal HA antibody (1:1,000; 90 min) and 2°(secondary) biotinconjugated goat anti-mouse IgG (1:200; 90 min) followed by streptavidin conjugated to Alexa-488 (1:500) for 90 min. Each labeling step was followed by 3-5 washes with 1% BSA (5 min each) to remove unbound Ab. All steps were performed at 4°C to prevent endocytosis of the channel. Following cell surface labeling, the cells were again washed in ice-cold PBS, fixed with 2% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/2% paraformaldehyde/PBS, blocked with 1% BSA and 10% goat serum as above, and then intracellular localized HA-hIK1 was labeled sequentially with 1°monoclonal HA antibody (1:1,000; 90 min), and 2°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. 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 ϫ 1024 pixels, using sequential 2-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, combined into a single figure, and RGB brightness/ contrast adjusted identically for all panels.
Immunoprecipitation (IP)-C59 constructs were translated from cDNA in the presence of [ 35 S]methionine using the TNT T7-Coupled Reticulocyte Lysate System (Promega, Madison, WI). Aliquots of the translation reaction were diluted 10-fold in IP buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% v/v Triton X-100, 1 mM EDTA containing complete EDTA-free protease inhibitor mixture mix), and pre-cleared with protein G (Omnisorb; Calbiochem). Samples were then incubated with either anti-V5 or anti-XP (1:5,000 dilution) antibodies, and immune complexes were precipitated by incubation with protein G. Following extensive washing, the protein G pellets were solubilized in Laemmli sample buffer and heated to 37°C for 5 min prior to removal of aggregates by centrifugation. Samples were resolved by SDS-PAGE (10% gel), and the gels were dried and subjected to autoradiography.
For co-immunoprecipitation of HA-and Myc-tagged hIK1 constructs, HEK293 cells were transiently transfected in 60-mm dishes using Li-pofectAMINE 2000 and 5 g of each plasmid (total, 10 g of DNA and 20 l of lipid). When only a single construct was transfected (HA or Myc), empty pcDNA3.1(ϩ) was included (5 g) to keep the final concentration of plasmid and lipid the same in all dishes. 18 -24 h posttransfection, cells were washed three times with ice-cold PBS and then lysed with IP buffer. Protein concentrations were determined and normalized to achieve equivalent loading. Crude lysates were then precleared with protein A-Sepharose beads (Sigma) and incubated with rabbit polyclonal anti-HA antibodies. Immune complexes were precipitated with protein A-Sepharose beads, followed by sequential washes in IP buffer containing 500, 300, and 150 mM (2ϫ) NaCl, supplemented with 1ϫ radioimmunoprecipitation assay (RIPA) 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 CS-IP, cells were grown to confluence in a 100-mm dish and then washed in ice-cold PBS, blocked in 1% BSA/PBS, and labeled with polyclonal HA.11 Ab (1:500) for 90 min at 4°C. Unbound Ab was removed by extensive washing in 1% BSA followed by washes in PBS. As above, all steps were performed at 4°C to prevent endocytosis of the channel and/or Ab. The cells were then lysed, and protein concentrations were normalized and the immune complexes directly subjected to IP as detailed above. Following transfer to nitrocellulose, an IB was performed using monoclonal HA Ab (1:1,000) as detailed below. In addition to the IP, 15 g of protein was set aside following cell lysis for an 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.
Proteinase K Digestion-Proteinase K digestion was performed following methods described previously (22). Briefly, HEK cells stably transfected with either wild-type or ZIP3,4 rSK3 were washed with ice-cold PBS and incubated with 150 mM NaCl, 2 mM CaCl 2 , 10 mM HEPES, pH 7.4, with or without 200 g/ml proteinase K (Sigma) at 37°C for 30 min. Proteinase K digestion was quenched by adding ice-cold PBS containing 6 mM phenylmethylsulfonyl fluoride and 25 mM EDTA. This treatment was followed by three washes in ice-cold PBS. Cleared lysates were prepared and analyzed by immunoblotting, as described above.
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 (15). Both DCEBIO and clotrimazole were made as 10,000-fold stock solutions in Me 2 SO. Complete EDTA-free protease inhibitor mixture mix was obtained from Roche Molecular Biochemicals.
Statistics-All data are presented as means Ϯ S.E., where n indicates the number of experiments. Statistical analysis was performed using a Student's t test. A value of p Ͻ 0.05 was considered statistically significant and is reported. All IF labeling and protein biochemical experiments were carried out a minimum of 3 times on each construct to ensure the voracity of our results.

RESULTS
We previously demonstrated that truncation of hIK1 at Lys 402 resulted in a complete loss of functional channels at the cell surface (20). This result could be caused by either a failure of channels to correctly traffic to the plasma membrane or from channels that traffic normally but are non-functional. To distinguish between these possibilities, an HA epitope was inserted into the extracellular loop between S3 and S4 (see "Experimental Procedures") such that cell surface expression could be evaluated by IF and CS-IP techniques, whereas function was assessed by the whole-cell patch clamp technique. Initially, we confirmed that HA-hIK1 could be detected at the cell surface by IF. As shown in Fig. 1A, wild-type HA-hIK1 is highly expressed at the cell surface (green) as well as being expressed intracellularly (red), as expected. Cells expressing hIK1 (no HA epitope) exhibited no fluorescence when labeled using an identical protocol (data not shown). We next determined whether insertion of the HA epitope had any effect on the biophysical and pharmacological characteristics of hIK1 as assessed by the excised, inside-out patch clamp technique. We and others demonstrated previously that hIK1 is activated by the pharmacological agent, DCEBIO (15), and inhibited by clotrimazole (2,4,7,11). In excised patch clamp studies, DCEBIO activated HA-hIK1 with an apparent K s of 3.8 Ϯ 0.8 M (n ϭ 3), whereas clotrimazole inhibited HA-hIK1 with an apparent K i of 65 Ϯ 5 nM (n ϭ 3), values similar to those reported previously for hIK1 (2,4,7,11,15). In addition, insertion of the HA epitope had no significant effect on either the single channel I-V relationship of the channel (chord conductance of 31 Ϯ 1 pS at Ϫ100 mV and 11 Ϯ 1 pS at ϩ100 mV, n ϭ 4) or Ca 2ϩ -dependent gating (K s ϭ 831 Ϯ 29 nM, Hill coefficient of 2.1; n ϭ 3), as these are values similar to what we and others have reported previously (7,11,12,14,23,24) for endogenously and heterologously expressed hIK1. In total, these results indicate that insertion of an HA epitope into hIK1 did not affect channel function. Thus, we utilized this construct to define the role of the cytoplasmic C terminus in the cell surface expression of hIK1.
Based on the observation that the HA epitope did not alter the function of hIK1 channel activity in excised patches, we confirmed HA-hIK1 could be activated by the pharmacological opener, DCEBIO (10 M), and inhibited by clotrimazole (3 M) in whole-cell patch clamp studies. Following establishment of the whole-cell configuration, current averaged 224.0 Ϯ 38.5 pA at Ϫ20 mV, indicative of very few active channels (Fig. 1E). DCEBIO increased the whole-cell current to 5,013.4 Ϯ 597.0 pA, and this was inhibited by clotrimazole to 930.5 Ϯ 176.4 (n ϭ 19). As the average capacitance for these 19 cells was 26.6 Ϯ 1.4 pF, this yielded an average current density of 166.6 Ϯ 19.4 pA/pF, as shown in Fig. 1E. Furthermore, as seen in Fig. 1D, HA-hIK1 displays no significant time-or voltagedependent activation during whole-cell recording as reported previously for hIK1 (11,12).
Truncation of hIK1 C Terminus Compromises Cell Surface Expression-We demonstrated previously (20) that functional hIK1 channels could be recorded in excised, inside-out patches following truncation at Leu 414 , whereas truncation at Lys 402 resulted in a complete loss of channel function. Thus, we initially introduced these stop mutations into HA-hIK1 to determine whether K402STOP and L414STOP traffic to the plasma membrane. As shown in Fig. 1, in contrast to HA-hIK1 (A), K402STOP (C) failed to express at the cell surface as assessed by IF (no green), although intracellular channel is prevalent in an intracellular compartment (red). In contrast, deletion of the last 14 amino acids (L414STOP) did not abrogate cell surface expression of the channel (Fig. 1B). To provide a quantitative estimate of cell surface expression of these truncated channels, we utilized the whole-cell patch clamp technique. Representative whole-cell recordings for HA-hIK1, K402STOP, and L414STOP are shown in Fig. 1D with average current density data shown in Fig. 1E. Whereas HA-hIK1 was highly expressed (166.6 Ϯ 19.4 pA/pF, n ϭ 19), K402STOP failed to express functional channels at the cell surface (0.6 Ϯ 0.2 pA/pF, n ϭ 12). In contrast to this complete loss of functional expression, L414STOP resulted in a more modest decrease in current density (95.8 Ϯ 20.8 pA/pF, n ϭ 17). These data suggest that amino acid residues within the distal C terminus, between Lys 402 and Leu 414 , are critical for the correct trafficking of hIK1 to the plasma membrane.
Mutation of the C-terminal Leucine Zipper Abrogates Membrane Trafficking of hIK1-Sequence gazing of the amino acids between Lys 402 and Leu 414 reveals two potential structural motifs that may be required for correct trafficking of hIK1: (i) a di-leucine motif (Leu 409 /Leu 410 ), and (ii) the terminal leucine (Leu 406 ) of a proposed leucine zipper motif. The last 59 amino acids of the hIK1 C terminus (Val 369 -Lys 427 ), encompassing the entire leucine zipper, are shown in Fig. 2. Di-leucine motifs are known to play a critical role in both Golgi exit as well as endocytic recycling of a wide range of proteins (25), including ion channels (26), whereas leucine zippers are important in protein-protein interactions (27). Therefore, we used site-directed mutagenesis to define the role of the di-leucine and leucine zipper motifs in the trafficking of hIK1. As shown in Fig. 3, mutation of Leu 409 /Leu 410 to alanines (DI-LEU) did not prevent trafficking of HA-hIK1 to the cell surface. Whole-cell patch clamp analysis confirmed functional expression of di-leucine at the cell surface (Fig.  4B), although the current density was significantly reduced (87.0 Ϯ 26.1 pA/pF; n ϭ 18) compared with HA-hIK1. Although these results suggest this di-leucine motif may play some role in the trafficking of hIK1, they cannot explain the complete loss of surface expression observed following truncation at Lys 402 (Fig. 1).
To assess the role of the leucine zipper in the trafficking of HA-hIK1, we initially mutated the 4th (Leu 399 ) and 5th (Leu 406 ) positions in the leucine zipper to proline (L399P) and phenylalanine (L406F). This mutation resulted in a complete abrogation of plasma membrane expression, as assessed by both IF (Fig. 3A, ZIP4,5) and cell surface immunoprecipitation (CS-IP, Fig. 3B). However, as shown in Fig. 3, this loss of function was not due to a loss of protein, as shown by both the IF labeling of intracellular channel (Fig. 3A) as well as the similar levels of protein upon immunoblot (Fig. 3B). Note that hIK1 runs as a doublet under the conditions utilized in our immunoblot and immunoprecipitation studies. Although the reasons for this micro-heterogeneity are unclear, it is a consistent finding for all of the constructs studied (Fig. 3B) and is also observed following in vitro translation in the presence of [ 35 S]methionine (data not shown). We confirmed the role of this C-terminal leucine zipper in the trafficking of HA-hIK1 by introducing additional double mutations in the 2nd and 3rd leucines (L385A/L392A; ZIP2,3), the 3rd and 5th leucines (L392A/L406A; ZIP3,5), and the 1st and 2nd leucines (L378A/ L385A; ZIP1,2). As shown in Fig. 3, ZIP2,3 and ZIP3,5 failed to traffic to the plasma membrane, although the channel was expressed intracellularly. In contrast, the ZIP1,2 mutation was clearly expressed at the cell surface as assessed by both IF (not shown) and CS-IP (Fig. 3B).
In coiled-coil domains, the hydrophobic pocket is determined by both the leucines in the d position, as well as hydrophobic amino acids in the a position (27). Thus, we determined whether the a position amino acids corresponding to Leu 399 and Leu 406 would similarly affect channel localization. Therefore, we mutated Ile 396 and Leu 403 to alanines (a-POS). As was true with the ZIP4,5 mutation, mutation of I396A/L403A resulted in a channel that failed to traffic to the plasma membrane as determined by both IF (Fig. 3A) and CS-IP (Fig. 3B). In an additional series of studies, we determined whether individual Leu/Ala mutations in the leucine zipper would affect trafficking of HA-hIK1. As shown in Fig. 3, A and B, mutating each of the leucine residues within the leucine zipper individually failed to affect surface expression of HA-hIK1.
To confirm the functional expression of these leucines zipper mutations, we utilized the whole-cell patch clamp technique to determine current densities for each of these constructs. As shown in Fig. 4, ZIP4,5P failed to express functional channels at the cell surface (0.2 Ϯ 0.1 pA/pF, n ϭ 14), consistent with our IF and CS-IP data. As this mutation (L399P/L406F) would be expected to cause a significant alteration in protein structure, we also generated an alanine-substituted mutant (L399A/ L406A; ZIP4,5A). As shown in Fig. 4B, this resulted in a similar reduction in current density Rescue of a Leucine Zipper Mutant by Lowering Temperature-Misfolded proteins are often retained in the endoplasmic reticulum. Our results indicate that hIK1 is retained within an intracellular compartment when the leucine zipper is mutated (Fig. 3A). Recent studies demonstrate that misfolded proteins, including cystic fibrosis transmembrane conductance regulator (28) and the HERG K ϩ channel (29 -32), will escape the endoplasmic reticulum if the cells are incubated at reduced temperatures. Therefore, we examined the effect of reducing the incubation temperature of cells from 37 to 27°C on plasma membrane expression and function of ZIP4,5A-HA-hIK1. As shown in Fig. 5A, reducing the temperature to 27°C resulted in a partial restoration of plasma membrane expression of Zip4,5A-HA-hIK1 (green, right panel). Note that we did not label the intracellular channel following incubation at 27°C in order to highlight expression at the cell surface. This partial correction of cell surface expression was confirmed by CS-IP measurements. As shown in Fig. 5B, Zip4,5A-HA-hIK1 was not detected at the cell surface when grown at 37°C, although protein expression was not compromised (IB in Fig. 5B). However, growing the cells at 27°C for 24 h resulted in a partial restoration of cell surface expression. Whole-cell patch clamp studies (Fig. 5, C and D) confirmed that, following incubation at 27°C for 24 h, DCEBIO stimulated a significant CLT-sensitive whole-cell current in ZIP4,5A-HA-hIK1 expressing cells compared with cells grown at 37°C (27°C, 46.9 Ϯ 13.5 pA/pF, n ϭ 17; 37°C, 4.8 Ϯ 2.4 pA/pF, n ϭ 10). Note that this channel displayed normal macroscopic gating kinetics following correction of the trafficking defect by incubation at 27°C suggesting this leucine zipper does not play a fundamental role in channel gating.
Mutation of the C-terminal Leucine Zipper Does Not Affect Assembly of hIK1 Subunits-The inability of HA-hIK1 to correctly traffic to the plasma membrane following mutation of the C-terminal leucine zipper could be caused by either a gross structural change, thereby precluding tetramer formation, or by a more subtle effect that affects only the distal C terminus of hIK1. To investigate the role of the C-terminal leucine zipper in tetramer formation, we generated a myc-tagged hIK1 and performed co-immunoprecipitation experiments. HA-hIK1 and myc-hIK1 were transiently transfected either alone or in combination into HEK293 cells, immunoprecipitated using anti-HA Ab, separated by SDS-PAGE, and immunoblotted with anti-myc Ab. As shown in Fig. 6, we were able to co-immuno- precipitate myc-hIK1 with an HA antibody (3rd lane), confirming assembly of hIK1 into minimally dimers and likely tetramers. Following mutation of the C-terminal leucine zipper in the HA-hIK1 backbone (ZIP4,5A-hIK1), a co-immunoprecipitate pulled down quantitatively similar amounts of Myc-tagged wild-type hIK1 (5th lane), suggesting that a heterotetrameric complex between wild-type and mutated subunits assembles correctly. Finally, when the leucine zipper was mutated in both myc-hIK1 and HA-hIK1, quantitatively similar amounts were also detected on co-immunoprecipitates (6th lane) demonstrating that mutation of the C-terminal leucine zipper does not affect channel tetramerization. representative examples of DCEBIO-stimulated whole-cell currents elicited by applying voltage pulses from Ϫ100 to ϩ80 mV in 20-mV increments for 250 ms every 2 s, from a holding potential of Ϫ60 mV. D, current density (pA/pF) for each construct at Ϫ20 mV (mean Ϯ S.E.; number of experiments is indicated in parentheses). hIK1 currents that were significantly different from wild-type hIK1 are indicated (*, p Ͻ 0.05, Student's t test).

Assembly of the Distal C Terminus of hIK1 Is Dependent
Upon an Intact Leucine Zipper-Our co-immunoprecipitation studies demonstrate that the C-terminal leucine zipper of hIK1 is not required for channel tetramerization. Thus, we considered the possibility that leucine zipper mutations might modify more subtle sub-domain interactions within the C terminus. To address this question we utilized a differentially epitopetagged (either V5 or Xpress) 59-amino acid C-terminal domain of hIK1 ( Fig. 2; Val 369 -Lys 427 ; C59), encompassing the leucine zipper, but not the calmodulin-binding domain, to examine the role of the leucine zipper in C-terminal self-assembly. As shown in Fig. 7A, following in vitro translation in the presence of [ 35 S]methionine and IP, the V5 and Xpress (XP) epitope-tagged constructs run at different apparent molecular masses of ϳ11 and 10 kDa, respectively (lanes 1 and 4), thereby allowing co-assembly to be evaluated via IP. Upon co-translation of wild-type V5 and XP constructs, V5 and XP antibodies pulled down products corresponding to both V5-C59 and XP-C59 (lanes 2 and 3, respectively), demonstrating co-assembly of these C-terminal domains. However, when the Xpress epitopetagged C-terminal leucine zipper was mutated (L399A/L406A; XP-ZIP4,5), co-assembly with V5-C59 was abrogated (lanes 6 and 7), demonstrating a critical role for the leucine zipper in C-terminal self-assembly. Interestingly, mutation of either L399A (XP-ZIP4; lanes 8 and 9) or L406A (XP-ZIP5; lanes 10 and 11) alone was not sufficient to disrupt co-assembly of this C-terminal domain. Thus, our results with the C terminus of hIK1 exactly mirror our results on full-length hIK1, i.e. double leucine zipper mutations are required to disrupt assembly/ trafficking. The specificity of these antibodies was confirmed by demonstrating that V5 Ab failed to pull down XP-C59 (lane 12) and XP Ab failed to pull down V5-C59 (lane 13) when either V5or XP-C59 was expressed alone. Furthermore, as shown in Fig.  7B, co-assembly of epitope-tagged C59 fragments occurred only when the fragments were translated together (co; 1st and 3rd lanes) and not when the fragments had been synthesized separately and then mixed afterward (mix; 2nd and 4th lanes).
The C-terminal Leucine Zipper Is Not Required for Membrane Trafficking of rSK3-hIK1 belongs to a gene family, KCNN, containing three additional members, the small conductance, apamin-sensitive, Ca 2ϩ -dependent K ϩ channels, SK1-3. Whereas hIK1 shares only 40 -42% identity with the SK channels, the C-terminal leucine zipper is conserved in SK1 and SK3 (the 2nd leucine position is replaced by phenylalanine in SK2). Thus, we determined whether the function of this leucine zipper as a trafficking determinant was conserved in rSK3. For these studies we mutated the 3rd and 4th leucines (L660A/L667A) of the C-terminal leucine zipper in rSK3 to alanines. Although the leucine zipper is conserved between hIK1 and rSK3, it is out of register by a single heptad repeat when sequence alignments are compared. Thus, the 3rd and 4th leucines of rSK3 correspond to the 4th and 5th leucines of hIK1. Cell surface expression and function were determined by proteinase K digestion and whole-cell patch clamp studies, respectively. Stable cell lines expressing either rSK3 or the mutant rSK3-ZIP3,4 were treated with proteinase K, a nonspecific serine protease that when applied externally cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. Cell lysates prepared from proteinase K-treated and control cells were then analyzed by SDS-PAGE and immunoblotting using a commercially available rSK3 antibody. As shown in Fig. 8A, lysates prepared from untreated cells expressing wild-type rSK3 (lane 1) and mutant rSK3-ZIP3,4 (lane 3) exhibit a single product with an apparent molecular mass of ϳ80 kDa, representative of full-length rSK3. Enzymatic digestion with externally applied proteinase K eliminated the bulk of the 80-kDa form and induced the appearance of a novel lower molecular mass product of ϳ50 kDa in both wild-type rSK3-(lane 2) and rSK3-ZIP3,4 (lane 4)-expressing cells. This lower molecular mass product is indicative of proteolytic digestion of rSK3. These data demonstrate that both wild-type rSK3 and mutant rSK3-ZIP3,4 are expressed predominantly on the cell surface. Functional expression of this mutated rSK3 channel was confirmed utilizing whole-cell patch clamp current density measurements. As shown in Fig.  8B, mutation of ZIP3,4 in rSK3 did not significantly affect current density (rSK3 ϭ 26.8 Ϯ 9.9 pA/pF, n ϭ 4; rSK3-ZIP3,4 ϭ 19.7 Ϯ 7.6 pA/pF, n ϭ 3). To further confirm that an intact leucine zipper is not required for efficient trafficking of rSK3, we introduced the additional double mutations, L667A/ L674A (rSK3-ZIP4,5) and L653A/L660A (rSK3-ZIP2,3), as these are the same mutations, with regard to leucine zipper position, made in hIK1. Cell surface expression was evaluated by proteinase K digestion. As shown in Fig. 8C, similar to the ZIP3,4 mutation, these additional leucine zipper mutations in rSK3 did not abrogate cell surface expression. Collectively these data suggest that, in contrast to hIK1, the C-terminal leucine zipper of rSK3 is not critical for correct trafficking to the plasma membrane. DISCUSSION Members of the KCNN gene family, including SK1-3 and IK1 are, as a family, widely expressed in both brain and peripheral tissues where they play critical roles in a host of physiological processes. However, the molecular motifs involved in assembly and trafficking of these channels to the plasma membrane, both in general and to specific subdomains (e.g. the basolateral membrane of epithelial cells), have been little evaluated. To begin to address these questions we inserted an HA epitope into the extracellular domain between S3 and S4 of hIK1. We demonstrate that this epitope insertion can be utilized for the detection of cell surface-localized hIK1 by both IF and cell surface IP techniques while having no effect on either the single channel properties (single channel conductance, Ca 2ϩ dependence), macroscopic gating kinetics, as assessed by whole-cell patch clamp, or pharmacological regulation (activated by DCEBIO, inhibited by clotrimazole) of hIK1. As such, we have utilized this construct to define the role of the distal C terminus in the trafficking of hIK1 to the plasma membrane.
Members of the KCNN gene family share a conserved leucine zipper motif in their distal C termini. Previous studies (33)(34)(35) have demonstrated a role for leucine zippers in the voltage-dependent gating of K ϩ and Ca 2ϩ channels. In contrast to these voltage-gated channels, mutation of the C-terminal leucine zipper in hIK1 had no apparent effect on macroscopic channel gating, i.e. following correction of the trafficking defect by incubating the cells at 27°C, the resultant DCEBIO-induced current displayed apparently normal gating characteristics (Fig. 5). Also, we demonstrate that an intact leucine zipper is not required for the assembly of hIK1; mutation of the Cterminal leucine zipper in all four subunits of hIK1 does not diminish their ability to assemble, as assessed by co-immunoprecipitation studies (Fig. 6). Rather, we demonstrate that an intact leucine zipper is required for the correct trafficking of hIK1 to the plasma membrane (Fig. 3), suggesting a more subtle folding defect as opposed to a complete loss of subunit assembly. The ability to correct this apparent folding defect by growing the cells at 27°C (Fig. 5) also argues for a more subtle folding effect. Our results are similar to data previously reported (36) for the GABA B receptor where the coiled-coil interaction of receptor subunit C termini stabilizes the active conformation but are not necessary for functional dimerization.
Although our studies have focused on the distal C terminus of hIK1, Joiner et al. (37) recently demonstrated that the assembly of calmodulin with the proximal C-terminal CAMBD is required for the targeting of hIK1 to the plasma membrane and that this was due to an enhanced assembly of hIK1 into tetramers in the presence of calmodulin. It was further demonstrated that the overexpression of the distal C terminus of hIK1, including the leucine zipper, inhibited the cell surface expression of full-length hIK1 (37). Here we demonstrate that the distal C terminus of hIK1 self-assembles in a leucine zipperdependent manner and that this is required for the correct targeting of hIK1 to the plasma membrane.
The small conductance Ca 2ϩ -activated K ϩ (SK) channels, SK1-3, share 42-44% identity with hIK1. Whereas the majority of this conservation occurs in the backbone (S1-S6) region of the channels, the C termini also demonstrate regions of high homology. Indeed, each of these channels possesses a CAMBD in their proximal C termini as well as a conserved leucine zipper in their distal C termini. Despite this conservation of a leucine zipper in SK1, SK3, and hIK1, our results demonstrate a lack of conservation in function for this motif, i.e. whereas mutation of the leucine zipper of hIK1 results in a dramatic loss of cell surface localized channel (Fig. 3), a similar mutation in SK3 had no effect on cell surface expression (Fig. 8) 35 S-labeled V5-and Xpress-tagged C termini were obtained by in vitro translation (translated constructs are indicated to the left) and subjected to immunoprecipitation using either anti-V5 (V5; lanes 1, 2, 7, 8, 11, and 12) or anti-Xpress antibodies (XP; lanes 3-6, 9, 10, and 13). The Ab used is indicated at the top of the lanes. Proteins were separated by SDS-PAGE followed by autoradiography. V5-C59 (lane 1) and XP-C59 (lane 4) ran at different apparent molecular masses as indicated by the arrows. Co-translation of V5-and XP-C59 resulted in both products being immunoprecipitated using either V5 Ab (lane 2) or XP Ab (lane 3), confirming co-assembly of this C-terminal fragment. Following mutation of ZIP4,5 in XP-C59 and co-translation with V5-C59, only a single product was pulled down using either XP Ab (lane 6) or V5 Ab (lane 7). In contrast, mutating either ZIP4 (lanes 8 and 9) or ZIP5 (lanes 10 and 11) alone did not inhibit co-assembly (note bands corresponding to both V5-and XP-C59). Lanes 12 and 13 demonstrate the specificity of the Abs utilized. B, 35 S-labeled V5-and Xpress-tagged C59 fragments were either co-translated (co) or translated individually then combined (mix). They were precipitated either with anti-V5 or with anti-Xpress antibodies, as described above. C59-hIK1 fragments only co-assembled (as shown by the doublet) when translated together, not when translated individually and then mixed. The experiments shown are representative of 3 separate experiments. that truncation of rSK2, distal to the CAMBD, did not affect expression of functional channels. In total, these results suggest that the distal C terminus in general, and the leucine zipper in particular, does not play a critical structural role in the SK members of the KCNN gene family, whereas it is absolutely required for the more distantly related IK gene family member.
Whereas our results clearly point to a role for the leucine zipper in the trafficking of hIK1, we further demonstrate that the most proximal leucine (Leu 378 , ZIP1) can be mutated, either alone or in combination with Leu 385 (ZIP1,2), with no deleterious effects on channel trafficking (Figs. 3 and 4). Interestingly, distinct studies have demonstrated that the CAMBD of IK and SK channels extends 95-98 amino acids from the S6 transmembrane domain (amino acids R287 to N384 in IK1) with the Ca 2ϩ -dependent binding domain being at the distal end of this motif (13,17). Thus, Leu 378 would be predicted to overlap with the Ca 2ϩ -dependent CAMBD such that it may not be accessible for protein-protein interactions.
We demonstrate that the distal C terminus of hIK1 (C59, Fig. 2) co-assembles in a leucine zipper-dependent manner (Fig. 7). Similar to the trafficking of full-length HA-hIK1, the co-assembly of C59 is only disrupted by the introduction of a double mutation (ZIP4,5), whereas single mutations (ZIP4 or ZIP5) have no effect on the assembly process. These results suggest that, in full-length hIK1, the distal C terminus selfassembles and that this is required for correct trafficking of the channel. Schumacher et al. (18) recently used x-ray crystallography to identify the structure of calmodulin bound to the proximal C terminus CAMBD of the rSK2 channel (18). Unfortunately, this crystal structure consists of only the CAMBD; it does not incorporate the C-terminal leucine zipper of rSK2. Therefore, it will be of particular interest to obtain crystals of the entire C terminus for both IK and SK channels and to determine the alignment of the leucine zipper ␣-helices within the context of the CAMBD-Ca 2ϩ -calmodulin complex so that we can envisage how mutations within the zipper compromise membrane trafficking of IK but not the SK channels.
Whereas the C-terminal leucine zipper of hIK1 may associate with itself there are at least two alternative possibilities that should be considered. First, leucine zippers are known to be involved in protein-protein interactions (27). Therefore, the C-terminal leucine zipper in hIK1 may be required for interactions with additional proteins necessary for the trafficking of the channel to the plasma membrane. In this regard, if the SK channels do not share these protein-protein interactions then mutation of the leucine zipper in these channels would not have the same effect on channel expression. A second possibility is that the C-terminal leucine zipper interacts with another domain within hIK1 itself. For example, hIK1 has a second potential leucine zipper extending from the cytosolic NH 3 terminus into the first transmembrane domain (Leu 18 /Leu 25 / Leu 32 /Leu 39 ), a domain not conserved in SK1-3. Thus, the NH 3 and C termini of hIK1 may assemble in order to form a channel that can traffic efficiently to the plasma membrane. This association of cytosolic domains is known to be important in the proper assembly and trafficking of other ion channels (38).
In conclusion, we demonstrate that the trafficking of hIK1 is dependent upon an intact C-terminal leucine zipper. Although this leucine zipper is not required for channel tetramerization, it appears to be crucial for the assembly of the C terminus of hIK1 into a trafficking competent conformation. Interestingly, FIG. 8. Mutation of the C-terminal leucine zipper in rSK3 does not inhibit expression of the channel at the plasma membrane. A, immunoblot of rSK3 and rSK3-Zip3,4 in stably expressing HEK293 cells in the absence (Ϫ) or following incubation in proteinase K (ϩ). Proteinase K digestion confirms that rSK3-Zip3,4 is expressed at the cell surface. B, average current density (pA/pF) measurements at ϩ40 mV for rSK3 and the L660A/L667A mutant (rSK3-ZIP3,4) (mean Ϯ S.E.; number of experiments is indicated in parentheses). Whole-cell patch clamp recording was performed in symmetrical K ϩ conditions, by applying voltage steps from Ϫ100 mV to ϩ80 mV in 20 mV increments for 250 ms every 2 s. The current densities for rSK3 and rSK3-ZIP3,4 were not significantly different. C, immunoblot of rSK3, rSK3-ZIP4,5, and rSK3-Zip2,3 in stably expressing HEK293 cells in the absence (Ϫ) or following incubation in proteinase K (ϩ). Proteinase K digestion confirms that rSK3-Zip4,5 and rSK3-ZIP2,3 are expressed at the cell surface. The immunoblots shown are representative of 3 separate experiments.