A Novel MaxiK Splice Variant Exhibits Dominant-negative Properties for Surface Expression*

Masoud M. ZareiDagger , Ning ZhuDagger , Abderrahmane AliouaDagger , Mansoureh EghbaliDagger , Enrico StefaniDagger §, and Ligia ToroDagger ||**

From the Departments of Dagger  Anesthesiology, § Physiology, and || Molecular and Medical Pharmacology and the  Brain Research Institute, UCLA, Los Angeles, California 90095-7115

Received for publication, September 28, 2000, and in revised form, February 2, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We identified a novel MaxiK alpha  subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha  (human SLO) and beta 1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha  and beta 1 subunits ~80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Voltage-dependent and Ca2+-activated K+ (MaxiK, BK) channels are expressed in a variety of tissues, including brain and smooth muscles (1, 2). Their functional properties and relative abundance vary from tissue to tissue and within the same tissue (1-5). The physiological role of MaxiK channels in different tissues is influenced not only by their single channel properties, but also by their expression level. In line with this view, they are strikingly abundant in smooth muscle, where they play a critical role in the control of vascular tone (6), and their expression level is exquisitely modulated during pregnancy (7). In brain, differential surface expression has been detected, suggesting a critical role of MaxiK channels in shaping the electrical activity in mammalian brain (8).

The elucidation of molecular mechanisms underlying MaxiK channel diversity has received much attention in recent years; however, those that determine channel expression levels in vertebrates are less understood. Vertebrate MaxiK channels are composed of the pore-forming alpha  and modulatory transmembrane beta  subunits (9-11). The alpha  subunit has seven transmembrane domains (S0-S6) (12) and seems to be the product of a single gene (13). In contrast, modulatory beta  subunits are products of several genes and contribute largely to the functional diversity of MaxiK channels. For instance, the beta 1 subunit can profoundly modulate the apparent voltage/Ca2+ sensitivity and activation/deactivation kinetics of the alpha  subunit (14-16). Functional diversity of MaxiK channels is further enhanced by alternative splicing of its alpha  (11) and beta  (17) subunits. Ten splice sites have been reported for the alpha  subunit; eight of them are located in the intracellular carboxyl terminus. However, only one splice insert has been reported to have profound functional effects on activation and deactivation kinetics and voltage/Ca2+ sensitivities of the vertebrate MaxiK channel (11, 18). Thus, at least two mechanisms can contribute to the functional diversity of MaxiK channels: association with various beta  subunits and splice variation. Recently, a Drosophila protein (DSLIP1) that reduces the number of functional channels in the plasma membrane has been isolated. This protein interacts with both human and Drosophila MaxiK channels (19). However, a mammalian DSLIP1 counterpart has not been described.

We now report a novel mammalian MaxiK channel alpha  subunit splice variant (SV1)1 that can regulate surface expression of both alpha  (hSLO) and beta 1 subunits of the human MaxiK channel when expressed in HEK293T cells. SV1 retains either alpha  or beta 1 subunits in the endoplasmic reticulum (ER). These findings suggest that SV1 may act as a dominant-negative expression regulator by inhibiting MaxiK alpha  and beta 1 subunit surface expression and show splice variation as a possible mechanism for regulation of MaxiK channel expression in mammalian tissues.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Biology

SV1 Isolation by RT-PCR-- Total RNA was extracted using 0.5~1.0 g of pregnant rat myometrium (Harlan Sprague-Dawley, 17 days pregnant) and the Totally RNA isolation kit (Ambion Inc.). Poly(A) RNA was purified using an Oligotex mRNA minikit (QIAGEN Inc.) according to the manufacturer's instructions. Approximately 100 ng of poly(A) RNA was converted to single-stranded cDNA by priming with an oligo(dT) primer, followed by a 20-µl reverse transcription reaction with 20 units of avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals).

Single-stranded cDNA was mixed with MaxiK gene-specific primers spanning a region between the linker of transmembrane segments S0 and S1 and transmembrane segment S2 (see Fig. 1A, arrows) and amplified with 3.5 units of Expand High Fidelity DNA polymerase (Roche Molecular Biochemicals) in a 100-µl reaction. The upstream primer anneals to nucleotides 419-437 (5'-AGGAGGTGGTGGCAGCCGA-3'), and the downstream primer anneals to nucleotides 652-672 (5'-AAACCGCAAGCCAAAGTAGAG-3') according to GenBankTM/EBI accession number U55995. PCR was performed by denaturing at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 7 min. Amplification products were separated on a 2% agarose gel and visualized by ethidium bromide staining. Sequences were determined using automated fluorescent sequencing (PerkinElmer Life Sciences).

Two amplification products were obtained: one with the expected size corresponding to the reported mammalian SLO sequences (253 base pairs) and another of larger size (352 base pairs). Sequence analysis showed that this product had an amino acid sequence identical to mammalian (human, rat, mice, bovine, and canine) SLO sequences, but contained an in-frame 33-amino acid segment. As expected for a splice site, the flanking nucleotides were Gs, which occur 60-80% of the time at splice sites (20). For functional analysis, the 33-amino acid segment was inserted into the human MaxiK alpha  subunit (hSLO); this construct was named splice variant 1 (SV1). Note that human and rat MaxiK channel alpha  subunits have an overall 99% amino acid identity.

RT-PCR Analysis-- RT-PCR was performed as described above using poly(A) RNA from rat brain and/or uterus (non-pregnant). The primers used to check the presence of the SV1 insert in rSLO were as follows (see Fig. 1C): Reaction 1 (Box B1), 5'-AAGATGGATGCGCTCATCATC-3' (forward primer, nucleotides 166-186, GenBankTM/EBI accession number U55995), which includes the start codon Met3 (21), and 5'-CATGAGGAGTCTAGGCATG-3' (reverse primer, SV1-specific; see Fig. 1B); and Reaction 2 (Box B2), 5'-GCATTTGAAAGATCCTCATTG-3' (forward primer, SV1-specific; see Fig. 1B) and 5'-CTCTATGATTTCAGGGACGTA-3' (reverse primer, nucleotides 1120-1140, GenBankTM/EBI accession number U55995). Detection of SV1 in brain and uterus was performed using the same SV1-specific primers. All products were confirmed by sequencing.

RNase Protection Assay (RPA)-- Probe DNAs were obtained by RT-PCR amplification using gene-specific primers for rSLO and SV1. To detect rSLO (generic sequence), we amplified a highly conserved region that includes the domain DQDDDD, known as the "Ca-bowl" (22), where no splicing sites have been reported. The upstream primer anneals to nucleotides 2842-2861 (5'-GGACCAAGACGATGATGATG-3'), and the downstream primer anneals to nucleotides 3060-3077 (5'-GCTAGTGTCTGCGGAGTG-3'). For SV1, the same specific primers used for RT-PCR analysis were used. Both amplification products were subcloned into the pCR2.1 vector (Invitrogen) and analyzed by nucleotide sequencing. Clones with the correct inserts were digested with EcoRV and KpnI and cloned into similarly digested pBluescript II KS vector (Stratagene). Plasmids were linearized with Acc65I, and digoxigenin-labeled antisense riboprobes were synthesized using the digoxigenin RNA labeling kit (Roche Molecular Biochemicals).

The RPA was performed using RNA isolated from non-pregnant rat myometrium and brain with the RNase protection kit (Roche Molecular Biochemicals). Each sample (100 µg of total RNA) was individually hybridized with 1 ng of rSLO probe or SV1 probe overnight at 45 °C. The unprotected single-stranded RNAs were digested with RNase A/T1. For each sample, total SV1-protected fragments and 1% of the rSLO-protected fragments were mixed, separated on an 8 M urea and 5% polyacrylamide gel, and transferred to positively charged nylon membranes (Roche Molecular Biochemicals) using contact blotting. Signals were detected by immunological and chemiluminescent detection with the chemiluminescent substrate CSPD® using the digoxigenin luminescent detection kit (Roche Molecular Biochemicals).

Constructs-- hSLO (12) and SV1 were tagged with the c-Myc epitope AEEQKLISEEDL at the N terminus. Tagged and untagged SV1 and hSLO behaved similarly. For simplicity, we refer to the c-Myc-tagged constructs as hSLO and SV1 throughout this work. hSLO-GFP and SV1-GFP were constructed by fusing GFP (pEGFP-N2, CLONTECH) to the C terminus of the human MaxiK alpha  subunit. N-terminal GFP tagging was avoided since it is known that the tag diminishes the voltage and Ca2+ sensitivities of the hSLO fusion protein (23). All constructs were subcloned into the pcDNA3 vector (Invitrogen). The GenBankTM/EBI accession numbers are U11058 for the human MaxiK pore-forming alpha  subunit and U25138 for the human MaxiK modulatory beta 1 subunit (KCNMB1).

Transfection

The calcium phosphate transfection protocol was used to transiently transfect HEK293T cells (24). Cells were grown to ~10% confluency and incubated for ~22 h with the transfection mixture. The transfection mixture was made by mixing (in a 1:1 ratio) a solution containing 250 mM CaCl2 and 20 ng/µl endotoxin-free plasmid with a buffer containing 42 mM HEPES, 274 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, and 11 mM dextrose, pH 7.05. This mixture was incubated for 20 min at room temperature prior to cell treatment. After transfection, cells were washed with phosphate-buffered saline (10 mM Na2HPO4, 2.3 mM NaH2PO4, 138 mM NaCl, and 2.7 mM KCl, pH 7.4) and fed with complete medium (Dulbecco's modified Eagle's medium, L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum; Life Technologies, Inc.). The day before immunocytochemistry experiments, cells were mechanically dissociated and transferred to chamber slides (Lab-Tek) that were precoated with 0.1 mg/ml poly-D-lysine and 0.1 mg/ml collagen. In experiments involving cotransfection of cDNAs, we used a 1:1 cDNA molar ratio.

Immunocytochemistry

Non-permeabilized Labeling (Live Cell Labeling)-- Three days after transfection, cells were incubated for 1 h at 37 °C with a 1:50 dilution of anti-c-Myc monoclonal antibody. Cells were washed and fixed for 30 min with 4% paraformaldehyde. After washing with phosphate-buffered saline, nonspecific binding was blocked by preincubating the cells with 5% donkey serum at room temperature. The excess serum was removed, and the secondary antibody (1:5000 dilution; Alexa 594, Molecular Probes, Inc.) was added for 1 h at room temperature. Cells were washed with phosphate-buffered saline, dried for 5 min, and mounted (Molecular Probe Prolong) for imaging.

Permeabilized Labeling-- Three days after transfection, cells were washed with phosphate-buffered saline and fixed for 30 min with 4% paraformaldehyde at room temperature. To permeabilize cells, subsequent solutions contained 0.2% Triton X-100 (Sigma). Nonspecific binding was blocked for 30 min at room temperature using 5% donkey serum. Primary antibodies were then added and incubated overnight at 4 °C. The next day, cells were washed and incubated for 1 h with the appropriate secondary antibodies at room temperature. The secondary antibodies were rhodamine red-X succinimidyl ester-conjugated donkey anti-rabbit or anti-mouse antibodies (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc.). Cells were washed again, and mounting was performed as described for non-permeabilized cells. Images were acquired by optically sectioning tissues every 0.5-1 µm with a confocal microscope (Leica) and analyzed using the Image-Pro Plus (Media Cybernetics) and Adobe Photoshop programs.

Antibodies-- Affinity-purified polyclonal antibodies were used against hSLO, Kv4.3 (Alomone Labs), and ERp72 (endoplasmic reticulum marker, Calbiochem) at 1:500, 1:500, and 1:1000 dilutions, respectively. These antibodies were raised against purified peptides corresponding to residues 883-896 (VNDTNVQFLDQDDD) of hSLO (7, 25), residues 451-467 ((Y)NEALELTGTPEEEH-Nle-GK) of Kv4.3 (26), and residues 623-638 ((C)FIDEHATKRSRTKEEL) of ERp72 protein (27, 28). To label the human beta 1 subunit, we used a polyclonal antibody (1:1000 dilution) raised against residues 85-102 (GRWAMLYHTEDTRDQNQQ) of KCNMB1 (29). Anti-c-Myc was a monoclonal antibody (1:50 dilution) raised against sequence AEEQKLISEEDLLRKRREQLKHKLEQLRNSCA (clone 9E10, Oncogene Science Inc.).

The lowest concentration of antibody that gives the highest signal was used in these studies. In all experiments, a negative control was performed by transfecting cells with no DNA and by preadsorbing each antibody with the corresponding antigenic peptide.

Electrophysiolgy

hSLO, hSLO-GFP, SV1, and SV1-GFP cDNAs were transiently expressed in HEK293T cells. One day after transfection, cells were plated on coverslips precoated with Cell-Tak (Collaborative Biomedical Products). Transfected cells were identified using a fluorescence microscope (for GFP constructs; CK40-RFL, Olympus) or by the CD8 method (30). In this method, CD8 cDNA is cotransfected with the cDNA of interest. Transfected cells are identified by light microscopy with the aid of anti-CD8 antibody-coated magnetic beads (Dynabeads®, Dynal, Inc.). In our experience, this method is highly successful when Shaker or hSLO K+ channels are cotransfected. Currents were measured 1-5 days after transfection using the cell-attached, inside-out, and whole-cell patch-clamp modes. Recording pipettes had resistances of ~2-5 megaohms and were filled with 105 mM potassium methanesulfonate, 5 mM KCl, 10 mM HEPES, 5 mM HEDTA, and 58 nM free Ca2+, pH 7.0. The bath solution was 105 mM potassium methanesulfonate, 5 mM KCl, 10 mM HEPES, and 10 µM free Ca2+, pH 7.0. A Ca2+ electrode was used to measure pCa (World Precision Instruments, Inc.) as previously described (31). Data was filtered at one-fourth the sampling frequency and acquired and analyzed with pCLAMP (Axon Instruments, Inc.). Normalized voltage activation curves of macroscopic currents were calculated as described previously (15). Values are expressed as means ± S.D. (n = number of experiments).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A Novel MaxiK Splice Variant-- A new splice variant of the MaxiK alpha  subunit (SV1) (Fig. 1A, gray portion) was isolated from rat myometrium by RT-PCR using MaxiK gene-specific primers spanning the S0-S1 linker and the middle of transmembrane segment S2 (arrows). Two PCR products were obtained (see "Experimental Procedures"): one with identical amino acid sequence to mammalian (human, rat, mice, bovine, and canine) SLO sequences and another containing an in-frame insert of 33 amino acids in the S1 transmembrane domain. Fig. 1B shows the nucleotide and amino acid sequences of the new insert and its position in transmembrane segment S1 (arrowhead). This insert contains a homologous sequence for the cAMP-dependent phosphorylation site (RXS*) (32). To discard the possibility of a cloning artifact, we amplified 447 bases of the 5'-end (including 3 bases upstream to the Met3 start codon) (21) and 708 bases of the 3'-end using pairs of SV1- and rSLO-specific primers. Using this strategy, only cDNAs containing SV1 sequences should be amplified. The diagram in Fig. 1C depicts the sizes of the expected products (1, 2) and the positions of the primers (arrows) and SV1 (gray boxes). The RT-PCRs yielded single bands (Fig. 1C) of the expected sizes (447 (lane 1) and 708 (lane 2) nucleotides) and sequences, indicating that indeed the SV1 insert is present in rat myometrium SLO cDNAs. As expected, when water (lane W) was added instead of cDNA, no bands were detected. RT-PCR using human myometrium cDNAs and the same primers also generated products with the expected sizes and sequences (data not shown).


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Fig. 1.   A new splice variant (SV1) at the N terminus of mammalian SLO. A, proposed SV1 topology shows that the insert elongates the linker between the S0 and S1 regions (gray dash line) in addition to substituting some of the S1 amino acids (gray portion of S1). Arrows mark the positions of RT-PCR primers used for initial isolation. B, shown are the nucleotide and amino acid sequences of the 33-amino acid splice insert. The arrowhead marks the location of the insert in the S1 transmembrane region (solid line) of rat or human SLO. The asterisk marks a potential cAMP-dependent phosphorylation site. C, shown is a bar representation of the SV1 5'- and 3'-amplified regions (upper panel). Arrows mark the positions and orientations of the primers. White boxes represent rSLO sequences, and gray boxes correspond to SV1 sequence. RT-PCR shows products of the expected sizes (lower panel). Lane 1, ~447 nucleotides (nt); lane 2, ~708 nucleotides; lane W, water used instead of cDNA as a control. D, RT-PCR (upper panel) and RPA (lower panel) show the presence of SV1 transcripts in uterus (lane U) and brain (lane B). RT-PCR with SV1 site-specific primers shows products of the expected sizes in uterus and brain, but no product in the control (lane W). RPA was carried out using an SV1-specific probe (99 nucleotides) and a rSLO-(2842-3078) generic probe (237 nucleotides) of a highly conserved sequence at the carboxyl terminus (see "Experimental Procedures"). Protected fragments of the expected sizes were present in both uterus (lane U) and brain (lane B). Only 1% of the generic protected fragment was loaded on the gel. E, hydrophobicity plots span the N terminus to the end of the S2 domain. The Genetics Computer Group program PEPPlOT, which uses Kyte and Doolittle hydropathy analysis (47), was used (averaging nine amino acids). The filled regions mark hydrophobic residues in stretches of approx 20 amino acids that correspond to transmembrane regions S0-S2. The solid bar marks the position of the insert (lower panel). F, the S1 hydrophobicity values of SV1 (dotted line) and hSLO (solid line) are almost identical.

The mRNA of the novel splice variant was detected in the uterus (Fig. 1D, lane U) and brain (lane B) using both RT-PCR (upper panel) and RPA (lower panel). RT-PCR was performed using SV1-specific primers that detected the expected 99-base pair product. SV1 transcripts were also detected by RPA. This method was used because of its much higher specificity and sensitivity compared with RT-PCR and Northern blotting. RPA analysis clearly demonstrated SV1 transcripts in uterus and also in brain (Fig. 1D). SV1-specific and rSLO-(2842-3078) generic probes distinguished two bands of the expected sizes. Signals for SV1 accounted for 1.2 ± 0.1% (n = 5) of the total rSLO message (note that only 1% of the total rSLO reaction was loaded; see "Experimental Procedures"). This low expression, together with the small size of the SV1-specific probe, likely contributed to the difficulty in detecting transcripts using Northern blotting (data not shown). Nevertheless, both the RT-PCR experiments (Fig. 1, C and D) and the RPA results confirm the authenticity of the SV1 splice variant and the existence of its mRNA in rat uterus and brain.

Because the SV1 insert is located within the hydrophobic S1 domain of SLO, we investigated if its insertion could impact the hydrophobic properties of S1. Fig. 1E illustrates the hydrophobicity plots of SLO (upper panel) and SV1 (lower panel) from the amino terminus to the end of S2; the location of the insert is marked with a solid bar. Note that rSLO and hSLO sequences are 100% conserved in this region and have 99% overall sequence identity. Striking is the similar hydrophobic pattern of S1 in both SLO and SV1 constructs. This similarity is further illustrated in Fig. 1F by overlapping S1 hydrophobicity values from SLO (solid line) and SV1 (dotted line). The high degree of overlap demonstrates that the overall hydrophobicity pattern of SLO is not disrupted by the splice insert in SV1, but rather it elongates the linker between S0 and S1.

SV1 Does Not Produce Detectable Ionic Currents in HEK293T Cells-- To investigate the functional impact of the SV1 splice insert, HEK293T cells were transfected with SV1 and hSLO constructs (Fig. 2; see "Experimental Procedures"). Currents were measured in cell-attached or inside-out configurations (Fig. 2, A-D, middle and right panels). Fig. 2A shows that cells transfected with SV1 (as assessed by coexpression of CD8; see "Experimental Procedures") had no detectable currents when measured in the cell-attached (n = 4) or inside-out (n = 4; 10 µM Ca2+) configurations. Changes in internal Ca2+ concentrations from 1.5 µM to 1.4 mM did not produce detectable currents. To ensure that patched cells were transfected with SV1, GFP was fused at the C terminus of SV1 (Fig. 2B, left panel). Similarly, SV1-GFP-transfected cells (as assessed by GFP fluorescence) showed no detectable currents in cell-attached (n = 12) or inside-out (n = 6) recordings in the presence of 10 µM intracellular Ca2+ (Fig. 2B). Furthermore, in whole-cell recordings, SV1- and SV1-GFP-transfected cells failed to generate any current (data not shown).


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Fig. 2.   SV1- or SV1-GFP-transfected HEK293T cells show no detectable current. Transfected cells were identified using the CD8 method (A and C) or the GFP fluorophore (B and D). Currents were elicited by holding cells at 0 mV and pulsing from -60 to +145 mV every 5 mV. No detectable current was measured from SV1 (A) or SV1-GFP (B) in cell-attached or inside-out recordings. In contrast, currents were detected from hSLO (C)- or hSLO-GFP (D)-transfected cells in cell-attached or inside-out configurations. In E, the fractional open probabilities (FPo) of hSLO (black-square) and hSLO-GFP () from inside-out recordings were plotted and fitted using a Boltzmann distribution. The calculated V0.5 values (the voltage at 50% FPo) in 10 µM internal Ca2+ were -30 ± 4 mV (n = 4) for hSLO and -35 ± 7 mV (n = 7) for hSLO-GFP. In F, V0.5 was plotted as a function of internal Ca2+ concentration. The Ca2+ sensitivities of hSLO (black-square) and hSLO-GFP () are practically the same. Values are the means of three to four experiments.

The lack of measurable currents from SV1 constructs was not caused by the addition of the c-Myc tag at the SV1 N terminus or fusion of GFP at its C terminus. SV1 constructs with (n = 8) or without (n = 12) c-Myc yielded no measurable currents. In addition, previous studies demonstrated that N-terminal c-Myc addition does not change the overall properties of the wild-type hSLO channels (12). We now demonstrate that the carboxyl-terminal GFP fusion does not change the overall voltage/Ca2+ sensitivities of hSLO compared with c-Myc-tagged hSLO. Fig. 2 (C and D) shows examples of currents in cell-attached or inside-out configurations. Voltage activation curves demonstrate that the voltage dependence of c-Myc-tagged hSLO (Fig. 2E, black-square) and that of GFP-tagged hSLO () were practically identical. Furthermore, current measurements at different Ca2+ concentrations indicated that the Ca2+ sensitivity of the hSLO-GFP channel was also similar (Fig. 2F). Because the fusion of GFP to the C terminus of hSLO does not interfere with its functional expression, it is likely that the addition of GFP to SV1 also keeps its intrinsic characteristics. Thus, our electrophysiological results using SV1-GFP demonstrate the inability of SV1 to produce detectable currents in the plasma membrane of HEK293T cells.

SV1 Displays No Surface Labeling-- To determine whether SV1 reaches the plasma membrane, we directly measured the cellular localization of SV1 and compared it with the hSLO expression pattern using fluorescent confocal microscopy. Since the N terminus of the MaxiK channel is positioned extracellularly (12), we used N-terminal c-Myc-tagged hSLO (Fig. 2C) or SV1 (Fig. 2A) and anti-c-Myc antibody to detect their surface expression in non-permeabilized cells (live cells). Fig. 3A clearly shows that hSLO was expressed in the plasma membrane (arrowheads) of HEK293T cells. The overlap of the fluorescent confocal image taken near the middle of the cells and the differential interference contrast (DIC) image show that the labeling coincided with the borders of the cells. Because cells were not permeabilized, no signal was detected inside the cells.


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Fig. 3.   Immunocytochemistry studies demonstrate the inability of SV1 to reach the surface membrane of HEK293T cells. A-C, confocal images of HEK293T cells transfected with c-Myc-tagged hSLO (A) or SV1 (B and C) and immunolabeled under non-permeabilized (A and B) or permeabilized (C) conditions. Anti-c-Myc antibody and Alexa 594 secondary antibody (red) were used. A, the overlap of DIC and hSLO (single confocal image) labeled cells shows the ability of hSLO to reach the plasma membrane (arrowheads). B, under identical conditions, SV1-transfected cells failed to show positive labeling (eight confocal images). C, after permeabilization, SV1-transfected cells display a perinuclear labeling pattern. D, counting the number of labeled cells/0.02 mm2 demonstrates that, under non-permeabilized conditions, SV1 could not be labeled because of its retention in the perinuclear region. Scale bars (A-C) = 20 µm.

In contrast to hSLO and under identical conditions, SV1-transfected cells showed no labeling even when the whole depth of the cells was scanned (Fig. 3B). This lack of labeling was observed in all studied cells in four different experiments. However, when cells were permeabilized, the anti-c-Myc antibody was able to reach its target inside the cell, and labeling of SV1 could be observed. The overlap of DIC and fluorescence images indicates that SV1 expression was concentrated in the perinuclear region of the cells (Fig. 3C).

Fig. 3D is the quantification of the number of labeled cells/field (0.02 mm2) in experiments as in Fig. 3 (A-C). In contrast to hSLO, SV1 showed no surface labeling; note that 40 different fields were examined. On the other hand, under permeabilized conditions, SV1 showed perinuclear labeling in every field examined, consistent with accumulation of protein in the ER.

MaxiK Splice Variant SV1 Is Localized to the Endoplasmic Reticulum-- The distribution of SV1 or SV1-GFP, seen at high magnification, showed a strong network expression pattern that spread within the cell bodies typical of ER localization. The overlap of DIC and fluorescent images (Fig. 4, A and B) illustrates the strong labeling around the nucleus (arrows) that extended outward in a network manner (arrowheads) without reaching the plasma membrane (asterisks).


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Fig. 4.   SV1 is localized to the ER. A and B, perinuclear distribution of both SV1 and SV1-GFP. Asterisks mark the plasma membrane as seen by DIC. Arrows mark the strong perinuclear staining, and arrowheads mark the mesh pattern typical of ER labeling. C, double-labeling experiments visualizing SV1 with GFP (green) and the ER with anti-ERp72 antibody (red). The high extent of colocalization was observed by overlapping all images (yellow). In C, 13 optical sections were overlapped to show the entire thickness of the cell. These images are representative of 47 (A), 37 (B), and 24 (C) cells examined in four to seven separate experiments. In this and the following figures, double-labeling experiments were visualized with rhodamine (red) and GFP (green) fluorophores. Scale bars = 10 µm.

To determine if the SV1 splice variant localizes in the ER, cells were transfected with SV1-GFP and double-labeled with an antibody against a resident ER protein, ERp72. SV1-GFP was used because no primary or secondary antibodies that can introduce background signals are needed; and thus, a more sensitive labeling is achieved. Anti-ERp72 antibody was selected because of ERp72 abundance and localization in the ER (27, 28). Fig. 4C illustrates SV1-GFP (green)-transfected cells that were permeabilized and labeled with anti-ERp72 antibody (red). The overlap of these images shows a clear colocalization of SV1-GFP and ERp72 proteins (yellow). These results indicate that SV1-GFP protein is synthesized, but is unable to leave the ER. Similar results were obtained in 24 cells in four separate experiments.

MaxiK Splice Variant SV1 Traps Coexpressed hSLO or beta 1 Subunits-- Tetramerization of Shaker K+ channels and assembly with the cytoplasmic Kv beta 2 subunit take place in the ER (33). In addition, increasing evidence indicates that beta  subunits play important roles in regulating ion channel surface expression. For example, cytoplasmic beta  subunits enhance plasma membrane expression of voltage-dependent K+ (34) and Ca2+ (35) channels, and the transmembrane beta  subunit SUR1, when properly assembled with its corresponding alpha  subunits (Kir6.1/2), masks retention signals located in both subunits, allowing surface expression of KATP channel complexes (36). Therefore, we investigated whether SV1 could interact with the alpha  (hSLO) or beta 1 subunits of MaxiK channels and serve as a dominant-negative regulator of channel expression, or if rescue of SV1 from the ER could occur.

The interaction of SV1-GFP with its counterpart hSLO or its transmembrane beta 1 subunit was studied in cotransfected cells under permeabilized conditions. We found that neither hSLO nor beta 1 could rescue SV1 from the ER; but instead, SV1 prevented the normal expression of both subunits in the plasma membrane. Fig. 5 compares the normal expression patterns of hSLO (A) and beta 1 (B) subunits when transfected alone with their expression when SV1-GFP was also expressed (C and D). Transfection with hSLO (43 cells, seven experiments) or beta 1 (56 cells, seven experiments) alone showed almost a uniform expression that covered the entire plasma membrane (Fig. 5, A and B, arrowheads). Perinuclear regions (arrows) had relatively higher expression in these cells, which can be explained by normal cell trafficking. This is in contrast to SV1 and SV1-GFP labeling, which concentrates near the nucleus, with no expression in the plasma membrane (Fig. 4, A and B). When SV1-GFP was coexpressed with hSLO, both proteins were found colocalized. Fig. 5C shows images of the same cell coexpressing SV1-GFP (assessed by GFP fluorescence; second panel) and hSLO (assessed by rhodamine labeling; third panel). Similarly, Fig. 5D shows that cotransfection of SV1-GFP with beta 1 induced its retention around the nucleus, consistent with ER retention. SV1-GFP expression was assessed by GFP fluorescence (green, second panel), and beta 1 expression in the same cell was assessed with rhodamine-conjugated secondary antibodies (red, third panel). The overlap images in Fig. 5 (C and D, fourth panels) show the high degree of SV1-GFP colocalization with hSLO or beta 1 subunits (yellow). Similar results were observed in 80 cells cotransfected with hSLO and 48 cells cotransfected with beta 1 (three experiments).


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Fig. 5.   SV1 traps coexpressed hSLO or beta 1 subunits in HEK293T cells. A and B show the normal expression patterns of MaxiK pore-forming alpha  (hSLO) and modulatory beta 1 subunits in permeabilized cells. Note that both subunits could express independently and readily reached the plasma membrane (arrowheads). The slightly higher labeling of the perinuclear region is likely due to normal trafficking (arrows). C shows that when SV1-GFP (green) and hSLO (red) were cotransfected, only a perinuclear labeling was observed for both proteins. hSLO was labeled with anti-c-Myc antibody. The overlap shows a high degree of colocalization in the perinuclear region (yellow), which did not extend to the plasma membrane (arrowheads). D shows a similar retention of the beta 1 subunit (red) by SV1-GFP and a high degree of colocalization (yellow). Arrowheads mark the outer boundaries of the cells. These results are representative of 43 (A), 56 (B), 80 (C), and 48 (D) cells examined in three to seven experiments. A and B are single 0.5-1-µm confocal sections; C and D are overlaps of eight confocal sections. Scale bars = 10 µm.

Our observations suggest that SV1-GFP may act as a dominant-negative expression regulator by trapping the MaxiK alpha  (hSLO) or beta 1 subunits in the ER. In addition, these results are consistent with the early coassembly of hSLO or beta 1 subunits with SV1-GFP in the ER.

MaxiK Splice Variant SV1 Allows the Coexpressed Kv4.3 Channel to Leave the ER and Does Not Interact with Cytosolic GFP-- To determine the specificity of the interaction of SV1 with hSLO or its beta 1 subunit, we cotransfected SV1 with Kv4.3 K+ channel or GFP cDNAs. Fig. 6A displays images of cells that were cotransfected with SV1-GFP and Kv4.3. Cells expressing SV1 were identified with GFP fluorescence (green), whereas cells expressing Kv4.3 were labeled with rhodamine-conjugated secondary antibodies (red). Note that not all cells in the field were expressing SV1-GFP (asterisks). Arrowheads in Fig. 6A point to the location of the outer cell membrane of a cell expressing both SV1-GFP and Kv4.3, as visualized by DIC. Note that SV1-GFP remained in the perinuclear region (arrows), whereas Kv4.3 was able to reach the plasma membrane (arrowheads). It is possible that Kv4.3 has a nonspecific interaction with SV1 since the level of labeling in the perinuclear region of cells expressing both Kv4.3 and SV1-GFP (arrows) was somewhat higher than in cells expressing Kv4.3 alone (asterisks). However, this possible interaction does not prevent Kv4.3 from reaching the plasma membrane.


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Fig. 6.   SV1 does not trap Kv4.3 in the ER and does not interact with GFP. A, HEK293T cells cotransfected with SV1-GFP and the Kv4.3 K+ channel. The image of SV1-GFP shows a single cell transfected with perinuclear distribution of SV1 (arrow). The Kv4.3 image shows, in the same field, several cells expressing Kv4.3 in their plasma membrane that were not cotransfected with SV1-GFP (asterisk). Arrows mark the perinuclear region of the cell coexpressing SV1-GFP and Kv4.3; note that Kv4.3 was able to reach the plasma membrane (arrowheads) (see overlap image). B, cells cotransfected with SV1 and GFP protein. SV1 labeling shows a clear perinuclear localization in two transfected cells (arrows). GFP fluorescence shows the expression of GFP alone in one cell (asterisk) and that coexpression with SV1 (arrows) did not prevent its diffuse expression away from the perinuclear region (arrowhead) (see overlap image). These images (eight confocal sections) are representative of 9 (A) and 12 (B) cells examined in three separate experiments. C, image analysis of experiments similar to those shown in A and B and in Fig. 5. The plot shows the ratio of signal intensity in the plasma membrane with respect to the perinuclear region in cells that were (black bars) or were not (gray bars; control) cotransfected with SV1 or SV1-GFP. Coexpression of SV1 with hSLO or beta 1 significantly (*) diminished expression of these MaxiK subunits in the plasma membrane. In contrast, expression of Kv4.3 or GFP was not altered in a significant manner. The numbers of cells examined were as follows: hSLO, 43; hSLO + SV1 or SV1-GFP, 80; beta 1, 56; beta 1 + SV1 or SV1-GFP, 48; Kv4.3, 4; Kv4.3 + SV1 or SV1-GFP, 9; GFP, 11; and GFP + SV1 or SV1-GFP, 12. Values are means ± S.E. Scale bars (A and B) = 10 µm.

Similar to Kv4.3, SV1 did not modify the GFP expression pattern (Fig. 6B). The asterisks mark a cell that was transfected only with GFP (no label in the SV1 image), whose expression was homogeneous throughout the cell. On the other hand, the arrows mark two cells that were cotransfected with SV1 and GFP; note that the expression pattern of GFP was unaltered in these cells. Moreover, we observed no significant interaction of GFP protein with SV1 in the perinuclear region.

Fig. 6C plots the intensity ratio of membrane to perinuclear labeling and demonstrates that cotransfection with SV1 (black bars) trapped both hSLO and beta 1 (*, p < 0.001), but had no significant effect on Kv4.3 and GFP expression. In summary, our results demonstrate that the MaxiK SV1 splice variant has dominant-negative properties when coexpressed with hSLO and its beta 1 subunit and that their interaction is specific.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We isolated a novel MaxiK splice variant (SV1) that contains a 33-amino acid insert between the S0 and S1 transmembrane domains of the alpha  subunit (Fig. 1, A and B). About six amino acids of the insert contributed to the S1 segment without affecting its hydrophobicity profile (Fig. 1, A and F). Even though the overall transmembrane hydrophobicity of hSLO did not change, the insert restrained the ability of SV1 to reach the plasma membrane of HEK293T cells. Our electrophysiological experiments (Fig. 2, A and B) and live cell labeling (Fig. 3B) demonstrate the inability of SV1 to produce any current or surface labeling, contrary to hSLO (Figs. 2, C and D; and 3A). In permeabilized cells, SV1 or SV1-GFP showed high expression around the nucleus and a weaker network expression within the cell bodies (Fig. 4, A and B), which corresponded to ER retention (Fig. 4C). In contrast, hSLO and beta 1 showed expression patterns that more uniformly covered the entire plasma membrane (Fig. 5, A and B). Taken together, our results demonstrate the inability of SV1 or SV1-GFP to reach the plasma membrane because of their retention in ER.

We also studied the interaction of the splice variant SV1 with hSLO, beta 1, Kv4.3, and GFP proteins. Our results demonstrate that the MaxiK splice variant trapped coexpressed hSLO and beta 1 in the ER (Fig. 5), but showed no effect on coexpressed GFP protein (Fig. 6). SV1-GFP seemed to have a limited nonspecific interaction with coexpressed Kv4.3 without inhibiting its surface expression (Fig. 6A, arrows). This interaction may be similar to the interactions seen between alpha  subunits of different classes of Kv potassium channels (37). In addition, the coexpression studies provide important insight into MaxiK subunit assembly and suggest the early assembly of hSLO and beta 1 subunits in the ER organelle. This is consistent with the early assembly of the alpha  subunit of K+ channels with cytosolic beta  subunits in the ER (33). It is also relevant to note that hSLO and beta 1 subunits could be expressed independently (Fig. 5) and that the beta 1 subunit did not seem to increase the functional expression of hSLO (data not shown), as is the case for the Kv beta 2 subunit and Shaker K+ channels (33).

SV1 and hSLO have identical amino acid sequences except for the 33-amino acid insert; however, one is retained in the ER (SV1), and the other is readily expressed in the plasma membrane (hSLO). The retention of SV1-GFP was not due to fragmented translation of SV1 since the C-terminal GFP domain of the SV1-GFP fusion protein was expressed. Also, it is unlikely that a thiol-mediated retention mechanism (38) could contribute to ER retention of SV1 since the insert is located in the cytosol and not in the lumen of the ER. Therefore, the two cysteine residues of the insert (Fig. 1B) are unable to interact with protein-disulfide isomerase located in ER lumen, which is involved in ER retention (38).

Another possible mechanism of ER retention may be caused by the inability of transport vesicles to form that would carry SV1 protein from ER to Golgi organelles. The cytosolic expression of the insert may require special assembly particles that would recognize the insert and include it in specific transport vesicles to deliver them to the Golgi and subsequently to the plasma membrane (39, 40). It is possible that, in HEK293T cells, this assembly particle(s) may be missing. This possibility was addressed by expressing SV1 in oocytes and polarized Madin-Darby canine kidney cells. SV1 produced no measurable currents in oocytes and no surface labeling in Madin-Darby canine kidney cells (data not shown).

Alternatively, SV1 may contain or unmask a novel ER retention signal. Typical retention signals are luminal KDEL and cytoplasmic KKXX motifs located proximal to the C terminus (41). None of these motifs are present in the SV1 insert, which is far away from the C terminus. However, other acidic retention signals containing arginines have been located in the N terminus of alternatively spliced variants of syntaxin-5 and the human invariant (Ii) chain of major histocompatibility complex class II (42, 43). Moreover, a new ER retention/retrieval signal (RKR) distant to both N and C termini has been identified that controls surface expression of fully assembled KATP channel complexes (36). Thus, it is possible that SV1 modulates expression of MaxiK channel complexes in a variety of tissues, allowing molecular plasticity and fulfillment of specific functions. It will be interesting to determine if SV1 can modulate the expression levels of MaxiK channels to different degrees, as is the case for Kv1.1 modulation of Kv1.2 and Kv1.4 (44), KvLQT1 isoform 2 of isoform 1 (45), or the HERG-A561V Long QT syndrome mutant of HERG K+ channel surface expression (46). Future studies will investigate SV1 effects on MaxiK channel expression in myometrium and brain cells.

In summary, we found a novel MaxiK channel splice variant (SV1) that acts as a dominant-negative regulator of MaxiK channel alpha  and beta 1 subunit expression. It is likely that SV1 induces differential surface expression of MaxiK channels and thus may contribute to plastic changes necessary for the proper function of excitable and non-excitable tissues. The role of SV1 as a dominant-negative regulator of MaxiK channel expression under physiological or pathological conditions needs to be determined. At present, we know that SV1 transcripts are present in myometrium and brain, and it is possible that their levels may change during the estrous cycle, pregnancy, or neurodevelopment or neurodegenerative conditions modifying MaxiK channel expression.

    ACKNOWLEDGEMENT

We thank Dr. Marcela Bravo-Zehnder for helpful discussions and experiments in Madin-Darby canine kidney cells.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL47382 (to L. T.) and GM52203 (to E. S.), a Human Frontier Science Program grant (to L. T.), and an American Heart Association Western States Affiliate postdoctoral fellowship (to A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Anesthesiology, UCLA, BH 509A CHS, P. O. Box 957115, Los Angeles, CA 90095-7115. Tel.: 310-794-7809; Fax: 310-825-5379; E-mail: ltoro@ucla.edu.

Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M008852200

    ABBREVIATIONS

The abbreviations used are: SV1, splice variant 1; hSLO, human SLO; rSLO, rat SLO; ER, endoplasmic reticulum; RT-PCR, reverse transcription-polymerase chain reaction; RPA, RNase protection assay; GFP, green fluorescent protein; DIC, differential interference contrast; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid.

    REFERENCES
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