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1-Subunits Increase Surface Expression of a Large-Conductance Ca²⁺-Activated K⁺ Channel Isoform

Department of Biology and Biochemistry, University of Houston, Houston, Texas

Submitted 4 January 2007; accepted in final form 18 February 2007

	ABSTRACT

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Auxiliary (beta) subunits of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels regulate the gating properties of the functional channel complex. Here we show that an avian 1-subunit also stimulates the trafficking of BK_Ca channels to the plasma membrane in HEK293T cells and in a native population of developing vertebrate neurons. One C-terminal variant of BK_Ca -subunits, called the VEDEC isoform after its five last residues, is largely retained in intracellular compartments when it is heterologously expressed in HEK293T cells. A closely related splice variant, called QEERL, shows high levels of constitutive trafficking to the plasma membrane. Co-expression of 1-subunits with the VEDEC isoform resulted in a large increase in surface BK_Ca channels as assessed by cell-surface biotinylation assays, whole cell recordings of membrane current, and confocal microscopy in HEK293T cells. Co-expression of 1-subunits slowed the gating kinetics of BK_Ca channels, as reported previously. Consistent with this, overexpression of 1-subunits in a native cell type that expresses intracellular VEDEC channels, embryonic day 9 chick ciliary ganglion neurons, resulted in a significant increase in macroscopic Ca²⁺-activated K⁺ current. Both the cytoplasmic N- and C-terminal domains of avian 1 are able to bind directly to VEDEC and QEERL channels. However, overexpression of the N-terminal domain by itself is sufficient to stimulate trafficking of VEDEC channels to the plasma membrane, whereas overexpression of either the cytoplasmic C-terminal domain or the extracellular loop domain did not affect surface expression of VEDEC.

	INTRODUCTION

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Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca channels) are expressed in a wide variety of excitable and nonexcitable tissues (Lu et al. 2006; Meredith et al. 2004). They are especially important in regulating the action potential waveform as well as the repetitive firing and oscillatory membrane properties of many neurons and neurosecretory cells (Cameron and Dryer 2000; Jovanovic et al. 2003; Lovell and McCobb 2001; Meredith et al. 2004; Shao et al. 1999; Thornelo et al. 2005; Werner et al. 2005).

As with other classes of ion channels, the pore-forming subunits of BK_Ca channels are often part of a larger complex that includes modulatory -subunits that modify the gating properties of associated -subunits but that are unable to form functional channels by themselves (Lu et al. 2006). The -subunits of BK_Ca channels are intrinsic membrane proteins containing cytoplasmic N- and C-terminal domains, and two transmembrane domains separated by a large extracellular linker (Knaus et al. 1996). In heterologous expression systems, co-expression of -subunits with a Slo1 -subunit alters the activation and deactivation kinetics, and the voltage and Ca²⁺ dependence of the resulting BK_Ca channels compared with those obtained when the Slo1 -subunits are expressed by themselves (Brenner et al. 2000; Cox and Aldrich 2000; Nimigean and Magleby 1999). A similar role in modulating gating properties has long been known for distinct classes of -subunits that interact with other types of K⁺ channels (Long et al. 2005; Morales et al. 1995; Rettig et al. 1994). Moreover, -subunits have also been shown to stimulate the trafficking of voltage-dependent K⁺ channels to the plasma membrane (Manganas et al. 2000; Shi et al. 1996; Uebele et al. 1996). The role of -subunits in regulating the trafficking of vertebrate BK_Ca channels has not been extensively studied. However Toro et al. (2006) observed that co-expression of a mammalian 1-subunit (also known as KCNMB1) can reduce steady-state cell surface expression of certain human Slo1 -subunits, an effect mediated in part by a putative endocytic signal in the cytoplasmic C-terminal of KCNMB1.

One feature that could obscure potential stimulatory effects of regulatory subunits on BK_Ca trafficking is that most of the 20 known Slo1 splice variants studied to date exhibit high constitutive expression on the plasma membrane. However, it has recently become clear that at least some Slo1 splice variants tend to be retained in intracellular compartments (Kwon and Guggino 2004; Wang et al. 2003; Zarei et al. 2001 2004). One such class of BK_Ca variants includes Slo1 channels with the motif VEDEC at the extreme C-terminal (Kim and Dryer, unpublished observations). These variants are strongly expressed in intracellular stores of developing chick ciliary ganglion neurons in which BK_Ca trafficking to the plasma membrane is a regulated process that requires stimulation by growth factors (Chae et al. 2005a,b). This VEDEC-containing splice variant is examined in detail here. For comparison, we also examine a second Slo1 variant that ends in the motif QEERL, which shows significantly higher constitutive surface expression.

The principle conclusion of the present study that an avian 1-subunit previously shown to modulate the gating properties of BK_Ca channels (Ramanathan et al. 1999, 2000) markedly stimulates trafficking of Slo1 -subunits to the plasma membrane in HEK293T cells. We further show that truncated 1-subunits composed of either the N- or C-terminal cytoplasmic domains are able to bind to co-expressed Slo1 -subunits. However, electrophysiological and biochemical experiments indicate that the N-terminal cytoplasmic domain of 1 is both necessary and sufficient to stimulate surface expression of at least some Slo1 -subunits on the plasma membrane.

	METHODS

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Plasmid constructs

Expression plasmids encoding N-terminal Myc-tagged VEDEC and QEERL isoforms of Slo1 were kindly provided by Dr. Min Li of the Department of Physiology at Johns Hopkins University. The primary sequences of the two isoforms examined in this study are shown in supplemental Fig. S1.¹ A full-length avian 1-subunit transcript (encoding a 200 amino acid protein) was isolated by RT-PCR from mRNA extracted from embryonic day 13 chick ciliary ganglion. This subunit is identical to the one originally isolated from chick fibroblasts (Oberst et al. 1997) and cochlear hair cells (Ramanathan et al. 1999) (GenBank/EMBL Accession No. O93393). Constructs encoding C-terminal green fluorescent protein (GFP)-tagged 1-subunits and truncated 1-subunits were generated by subcloning PCR products into pcDNA3.1/CT-GFP-TOPO vector (Invitrogen, Carlsbad, CA). The truncated subunits included a portion of the N-terminal cytoplasmic domain (residues 1–42; 1N-GFP), the extracellular loop domain (residues 40–155; 1L-GFP), and the C-terminal cytoplasmic domain (residues 175–200; 1C-GFP). We also prepared deletion constructs that lacked the last 45 residues (1C-GFP) or the first 40 residues (1N-GFP) of avian 1. Plasmids encoding glutathione S-transferase (GST)-1 fusion proteins, including GST-1N (residues 1–22), GST-1L (residues 40–155), and GST-1C (residues 175–200), were generated by PCR from full-length 1. The 5' and 3' oligonucleotides incorporated BamHI and EcoRI enzyme sites, respectively, to facilitate subcloning into pGEX-KG expression vector (Amersham Biosciences, Piscataway, NJ). A full-length avian 1-subunit (1-GFP) was used as the template for point mutations. The N-terminal point mutants (E14A, T15A, R16A, L20A, and Q21A) were made by the QuickChange II site-directed mutagenesis system (Stratagene, La Jolla, CA). The fidelity of all constructs was confirmed by sequencing.

Cell culture and transfection

HEK293T (human embryonic kidney) cells were grown in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a 5% CO₂ incubator. In most experiments, HEK293T cells were transiently transfected in 10-cm dishes (for biochemistry) or 24-well plates (for electrophysiology) using Lipofectamine-2000 (Invitrogen) in serum-reduced medium (Opti-MEM, Invitrogen) following the manufacturer's instructions. The DNA concentration in the transfection medium was 1 µg/ml of each plasmid. Ciliary ganglion neurons were transfected in 24-well plates using Lipofectamine-2000 with a final DNA concentration of 10 µg/ml of 1-GFP or GFP plasmid. Cells were used for physiology or biochemistry 24–48 h after transfection. Longer incubation times appeared to be optimal for biochemical investigations. HEK293T cells were transfected with high efficiency (70–80%) but the efficiency for ciliary ganglion neurons was very low (<3%), which precluded biochemical analysis of transfected cells in that system.

Electrophysiology

In experiments with HEK293T cells, plasmids encoding GFP or GFP-fusion proteins were co-transfected with either VEDEC or QEERL expression vectors, and whole cell recordings were made from fluorescent HEK293T cells using standard methods similar to those described previously (Chae et al. 2005a,b). However, in the present studies, the bathing solution contained (in mM) 150 NaCl, 0.08 KCl, 0.8 MgCl₂, 5.4 CaCl₂, 10 glucose, and 10 HEPES, and the pH was adjusted to 7.4 with NaOH. The pipette solution contained (in mM) 145 NaCl, 2 KCl, and 6.2 MgCl₂ and 5 µM CaCl₂, pH 7.2. The free concentration of Ca²⁺ in this solution was checked using an Orion 97–20 calcium electrode (Thermo Fisher Scientific, Waltham, MA) calibrated using commercial solution standards obtained from World Precision Instruments (Sarasota, FL). HEK293T cells do not express endogenous voltage-activated Ca²⁺ currents, and these ionic conditions were chosen to provide sufficient intracellular Ca²⁺ for activation of BK_Ca channels by depolarizing step pulses (Ramanathan et al. 2000) while keeping the resulting macroscopic currents small enough to avoid saturation of the patch-clamp amplifier or significant series resistance errors. The later were achieved by reducing the concentration of permeant ions by 60-fold while still maintaining a physiological E_K of –80 mV. Whole cell currents were not observed when recording pipettes contained 0 CaCl₂ and 10 mM EGTA (data not shown). Recording electrodes were made from thin borosilicate glass and fire-polished. They had resistances of 3–4 M when filled with pipette saline, and it was possible to compensate 85% of this without introducing oscillations into the current output of the patch-clamp amplifier (Axopatch 1D, Axon Instruments). All physiological experiments were conducted at room temperature. Currents in HEK293T cells were evoked by a series of eight 450-ms depolarizing steps (from –25 to +80 mV in 15-mV increments) from a holding potential of –40 mV. Currents were digitized and analyzed off-line using PClamp software v 9.1 (Axon Instruments). Rising phases of currents were fitted as single- or double-exponential curves by nonlinear least squares using the Levenberg-Marquarrdt algorithms in PClamp. Activation curves were constructed by plotting fractional activation (the normalized conductance G/G_max) against command potential and fitting the resulting curves with the Boltzmann function G/G_max = {1 + exp[–(V – V_1/2) qF/RT]}^–1.

where G is chord conductance at the command potential V assuming an E_K of –80 mV, G_max is the maximal conductance, V_1/2 is the voltage of half-maximal activation, q is a slope constant, and F, R, and T have their usual significance. These fits were done by nonlinear least squares using the Levenberg-Marquardt algorithms implemented in Origin v7.0 software (Northhampton, MA). Current voltage from the same data sets were fitted with spline curves that do not have theoretical significance. In some experiments, iberiotoxin (IbTx; Alamone, Jerusalem, Israel) was added to external salines and applied to transfected HEK293T cells at a final concentration of 10 nM 30 min prior to recording. Whole cell recordings from transfected chick ciliary ganglion neurons were done using methods described in detail in many previous reports (Cameron et al. 1998; Chae et al. 2005a,b; Lhuillier and Dryer 2002). Briefly, currents were evoked by step pulses in the presence and absence of external Ca²⁺ from a holding potential of –60 mV, and the amplitude of outward currents carried by BK_Ca was determined by digital subtraction (control–Ca²⁺-free). We have previously shown that all of the macroscopic Ca²⁺-dependent outward current detected by this protocol is carried by BK_Ca channels (Lhuillier and Dryer 2000).

Cell-surface biotinylation assays

HEK293T cells were grown on 100-mm plates for 24–48 h after transfection. Cell-surface biotinylation was carried out by treating intact cells with sulfo-N-hydroxy-succinimidobiotin (Pierce Biotechnology, Rockford, IL; 1 mg/ml in PBS buffer) for 1 h on ice with gentle shaking. Cold PBS buffer containing 100 mM glycine was then added to stop the reaction. After an additional 20 min of incubation, the cells were collected and lysed by gentle trituration in PBS containing 0.5% Triton X-100. The biotinylated proteins from the cell surface were recovered from the lysates by incubation with immobilized streptavidin-agarose beads (Pierce Biotechnology). Bound proteins were eluted from the beads in Laemli buffer, and a portion of the original lysate was also saved. Total proteins were quantified and then separated on SDS-PAGE, and proteins quantified by immunoblot analysis. These and all subsequent biochemical experiments were repeated at least three times.

Co-immunoprecipitation and immunoblotting

Co-immunoprecipitation and immunoblot analyses were performed as described previously (Zou et al. 2005). For co-immunoprecipitation, GFP-1, GFP-1N, GFP-1L, or GFP-1C in pcDNA3.1/CT-GFP were expressed in HEK293T cells, either alone or together with N-terminal Myc-tagged VEDEC or QEERL isoforms of Slo1. Cells were lysed in 50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, 1 mM PMSF, and protease inhibitor mixture (Sigma, St. Louis, MO). Cell extracts (500 µg of protein) were incubated in the presence of primary antibodies, including anti-GFP (Invitrogen, 1:1,000) or IgG (1–2 µg), for 4 h at 4°C, followed by the addition of 20 µl of protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 12 h. Pellets were collected by centrifugation and washed four times in PBS containing 0.5% Triton X-100 (PBST) and boiled for 5 min in 30 µl of Laemli sample buffer, and 15 µl of each sample were separated by SDS-PAGE on 10% gels. Cell extracted protein (100 µg) was used as control in each experiment. Proteins were transferred to nitrocellulose filters by wet transfer (1 h) on ice. Blots were blocked with 5% nonfat dried milk dissolved in TBST buffer (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature, washed three times with TBST buffer, incubated with the primary antibody (anti-c-Myc, 1:1,000) overnight at 4°C, and washed again with TBST, and the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000) for 2 h at room temperature. The proteins were visualized using a chemiluminescent substrate (Super Signal West Pico, Pierce Biotechnology).

GST pull-down assay

GST, GST-1N, GST-1L, or GST-1C fusion proteins were expressed and extracted from E. coli strain BL21, and 100 µg of each fusion protein was separately bound to 30 µl of glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions. HEK293T cells were transfected with Myc-tagged VEDEC or Myc-tagged QEERL, and lysed, and the soluble cell extracts (containing 500 µg of protein per sample) were added to the beads, and the samples were incubated overnight at 4°C with gentle rotation. The beads were washed three times with PBST and boiled for 5 min in 30 µl of Laemli sample buffer, and 15 µl of each sample was separated on 10% SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblot as described in the preceding text.

Confocal microscopy

For immunofluorescent labeling, HEK293T cells were grown on poly-D-lysine-coated glass coverslips, and cells were transiently transfected with Myc-tagged VEDEC or Myc-tagged QEERL expression vectors. Cells were subsequently fixed with 4% paraformaldehyde, blocked with 10% normal goat serum, and then incubated sequentially with anti-c-Myc antibody for 2–3 h (antibody 9B11, Cell Signaling Technology, Danvers, MA), and Alexa-488-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR) for 1–2 h. Plasma membranes were stained with Alexa Fluor 594-wheat germ agglutinin (Molecular Probes). After staining, the coverslips were washed in PBS and mounted using Vectashield (Vector Laboratories). Images were collected on an Olympus FV-1000 inverted stage confocal microscope using a Plan Apo N x60 1.42 NA oil-immersion objective. Green fluorescence was evoked using an excitation wavelength of 495 nm while monitoring emission at 519 nm. Red fluorescence was evoked by excitation at 580 nm and emission was monitored at 620 nm.

Statistics

All quantitative data are presented as means ± SE. The data in bar graphs were compiled from 9 to 25 cells in each group. Data were analyzed by one-way ANOVA followed by post hoc analysis (Statistica, Statsoft, Tulsa, OK) using Tukey's honest significant difference test for unequal sample size, with P < 0.05 regarded as significant.

	RESULTS

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C-terminal motifs affect surface expression of Slo1 isoforms in HEK 293T cells

In this study, we have examined two Slo1 -subunit splice variants that are identical until residue 1108, which is located in the extreme C-terminal of the channels (supplemental Fig. 1). We refer to the larger isoform as the VEDEC variant, and the smaller isoform as the QEERL variant, after the last five residues in each isoform. To examine surface expression of the two Slo1 isoforms, we transiently transfected HEK293T cells with tagged VEDEC and QEERL constructs. We observed that N-terminal (ectofacial) Myc-tagged VEDEC channels are largely retained within intracellular compartments in this expression system. In contrast, Myc-tagged QEERL channels are expressed at a substantially higher level on the cell surface (Fig. 1). The differences in the steady-state surface expression of the two splice variants can be visualized directly by confocal microscopy (Fig. 1A) and by means of cell-surface biotinylation assays (Fig. 1B) using anti-c-Myc antibodies to detect the tags. In confocal experiments, VEDEC channels, which give rise to a green fluorescent signal in these experiments, appear to be distributed throughout the intracellular space of the cells and appear to be excluded from the plasma membrane, which was labeled using wheat germ agglutinin (WGA) and which yields red fluorescence. In contrast, QEERL channels exhibit robust expression primarily in the periphery of the cell and especially in the plasma membrane (Fig. 1A). Consistent with this, we observed that only a small portion of the VEDEC protein can be biotinylated by a 1-h exposure to a membrane-impermeable reagent (sulfo-N-hydroxy-succinimidobiotin) in intact HEK293T cells, indicating that most of the VEDEC channels are in intracellular pools. However, a much larger portion of the QEERL channels can be biotinylated by this procedure (Fig. 1B), indicating that many of the QEERL channels in the cell periphery have reached the cell surface. It bears noting that the same c-Myc antibody was used to monitor the trafficking of both Slo1 isoforms in these experiments. The higher molecular-weight bands labeled in the immunoblot procedures are from Slo1 dimers (Wang et al. 2003).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Surface expression of two different isoforms of Slo1 expressed in HEK293T cells. A: confocal images showing that many of the Myc-tagged VEDEC channels (green) are localized in the interior of the cell and are generally excluded from the plasma membrane shown by staining with Alexa Fluor 594-conjugated wheat germ agglutinin (WGA; red). By contrast, Myc-tagged QEERL channels extensively co-localize with WGA in the plasma membrane. B: cell-surface biotinylation assay in which surface proteins are covalently modified by a membrane-impermeable biotinylation reagent applied to intact cells. The cells are then lysed and surface channels are isolated using streptavidin-agarose beads, and examined by immunoblot analysis (surface) using an antibody agaiast myc tag. The blots shown below (total) are myc signals in samples of the same cell lysates in which surface proteins have not been removed. The immunoblots show that most of the Myc-tagged VEDEC is intracellular, whereas a higher percentage of QEERL can be detected on the cell surface. Comparable levels of total protein are achieved with both isoforms. The higher molecular weight (MW; >200 kD) band in these immunoblots represents Slo1 dimer. C: examples of typical macroscopic recordings in transfected HEK293T cells. Cells expressing Myc-tagged VEDEC channels have modest outward currents evoked by a series of 8 450-ms depolarizing steps (from –25 mV to +80 mV in 15-mV increments) from a holding potential of –40 mV (protocol shown below current traces). By contrast, cells expressing Myc-tagged QEERL channels typically have much larger currents when subjected to the identical voltage-clamp protocol. Pipette solutions in these recordings contained 5 µM CaCl2. These current traces have not been leak-subtracted. D: summary of results from many cells. Bar graphs show means ± SE of currents evoked by a depolarizing step to +65 mV. In this and subsequent figures, data in each bar were from 9 to 25 cells. Currents carried by both channel isoforms are completely blocked by pretreatment with 10 nM iberiotoxin (IbTx). Asterisks indicate P < 0.05 compared with VEDEC (* and **) and P < 0.05 compared with QEERL (***) determined by ANOVA and a post hoc test.

Co-expression of the avian Slo 1-subunit of BK_Ca channels increases steady-state expression of the VEDEC isoform on the cell surface

The avian 1-subunit was cloned from embryonic day 13 chick ciliary ganglion neurons by RT-PCR and placed in an expression vector that allows expression of this protein with a C-terminal GFP tag. This auxiliary subunit is identical to the 1-subunit isolated from embryonic chick cochlear hair cells (Ramanathan et al. 1999). When the 1-subunit is expressed by itself in HEK293T cells, the GFP tag can be found throughout the cells (Fig. 6C) and there is no detectable voltage-evoked ionic current in whole cell recordings (Fig. 3B, left). We do not observe punctuate intracellular labeling pattern as reported for mammalian 1-subunits expressed by themselves (Toro et al. 2006). However, there is substantial expression of VEDEC and 1-GFP on the cell surface when the two subunits are co-expressed as determined by confocal microscopy (Fig. 2A, bottom). Note that in these experiments, the VEDEC channels give rise to red fluorescence because we used a different secondary antibody. In experiments to control for possible effects of the GFP tag, we observed that VEDEC channels remain largely intracellular in HEK293T cells co-expressing GFP (Fig. 2A, top). Consistent with this pattern, cell-surface biotinylation assays show an enhanced surface expression of VEDEC when it is co-expressed with the 1-subunit (Fig. 2B). Expression of 1-GFP does not affect the cell surface distribution of the QEERL form of Slo1 as revealed by confocal microscopy (data not shown) or cell-surface biotinylation assay (Fig. 2B).

View larger version (41K): [in this window] [in a new window]   FIG. 2. Co-expression of avian 1-subunits increases cell-surface expression of VEDEC channels. A: confocal images showing that Myc-tagged VEDEC channels (red) are primarily intracellular when they are co-expressed with green fluorescent protein (GFP, green). However, co-expression of 1-GFP causes the VEDEC channels to be primarily located at the periphery of the cells. B: similar pattern is observed in cell-surface biotinylation assays. Specifically, co-expression of 1 increases surface expression of VEDEC forms but has no effect on QEERL, which has much higher constitutive expression on the cell surface.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Co-expression of avian 1-subunits affects macroscopic currents. A: macroscopic currents in HEK293T cells expressing VEDEC and QEERL subunits along with full-length 1-GFP, as indicated. Voltage-clamp protocols are identical to those used in Fig. 1C. B: summary of results from many cells showing large increase in mean VEDEC-mediated current evoked by co-expression of 1-subunits (*). However, 1 does not cause a statistically significant increase in current carried by QEERL channels. No current is detected in cells expressing 1 channels by themselves, and treatment with 10 nM IbTX blocks all currents (**). C: co-expression of 1-subunits markedly slows activation kinetics as seen in the mean type to peak in currents evoked by a step to +25 mV. Left: traces are from typical cells in which the current amplitudes are differentially scaled and then superimposed to show effects of 1 on kinetics more clearly. , traces from cells co-expressing 1 along with the Slo1 isoforms. Right: bar graph shows time constant of a single-exponential fit to the activation of the currents evoked at +65 mV (±SE). Examples of currents with superimposed fitted curves with time constants of 4.76 ms (VEDEC) and 44.7 ms (VEDEC + 1) are shown above the bar graph. Slowed activation and deactivation occurs with both the VEDEC and QEERL forms. *, P < 0.05.

View larger version (29K): [in this window] [in a new window]   FIG. 4. The effect of 1 on VEDEC current amplitudes cannot be attributed to a modest shift to more negative command potentials for the activation of macroscopic currents. A: current-voltage diagram for VEDEC channels expressed alone and in combination with 1 as indicated (left) and plot of fractional activation (G/Gmax) against command potential derived from the same data set (right). Note increased current at all command potentials when 1-subunits are present. Right: plot is fitted with Boltzmann curves. B: similar plots for QEERL channels expressed alone and in combination with 1 as indicated. Data points are means ± SE from 5 cells in each group.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Exogenous overexpression of Slo 1-subunit increases mean Ca2+-dependent outward current in isolated E9 ciliary ganglion neurons. Cells were isolated, transfected, and used for electrophysiology 24 h later. A: representative macroscopic currents in E9 CG expressing GFP (top left) or full-length 1-GFP (top right) evoked by a series of 450-ms depolarizing pulses (–30 to +40 mV in 10-mV increments) from a holding potential of –60 mV. Step commands were evoked from the same cells in the presence and absence of external Ca2+, and the traces shown are net Ca2+-dependent currents from the same cells calculated by digital subtraction (control –Ca2+-free). The current-voltage relationship is shown for each of these families of currents (bottom left). The rising phase of the currents is slower in cells expressing 1-GFP (bottom right). These traces are a portion of the currents evoked by a step pulse to +40 mV shown above. They are plotted on a different vertical scale to facilitate comparison of the time course, along with superimposed fitted single- or double-exponential curves with time constants and relative weights as indicated. B: summary of results from 14 cells in each group showing larger mean Ca2+-depependent current in neurons overexpressing 1-subunits. Currents were isolated as described in A. *, P < 0.05.

View larger version (30K): [in this window] [in a new window]   FIG. 6. Binding of 1-subunits and 1 deletion mutants to VEDEC channels. A: schematic diagram of 1-GFP fusion proteins (left) and co-immunoprecipitation experiment showing binding of full-length 1-GFP to VEDEC channels in HEK293T cells (right). Full-length 1-GFP was immunoprecipitated using anti-GFP and associated proteins were analyzed by immunoblot using anti-Myc. B: Co-immunoprecipitation to analyze binding of 1 deletion mutants outlined in A to VEDEC. VEDEC can bind to 1C, 1N, and 1N (left) as well as to 1C (right). However, 1L does not appear to bind to VEDEC channels (right). C: expression of the deletion mutant proteins in HEK293T cells by visualization of the GFP tags.

One question that emerges is whether the avian 1-subunit can regulate surface expression of functional BK_Ca channels in the ciliary ganglion neurons, especially because that was the tissue source from which our 1 clone was isolated. We have previously shown that functional BK_Ca channels are maintained in intracellular pools within ciliary neurons prior to stimulation by appropriate growth factors (Cameron and Dryer 2000; Chae et al. 2005a,b; Lhuillier and Dryer 2002). However, cell surface trafficking in a subset of cells within the ganglion, the choroid neurons, can proceed in the absence of growth factor stimulation (Cameron and Dryer 2000). To examine this issue, E9 ciliary ganglion neurons were isolated and transfected with expression plasmids encoding either 1-GFP or GFP alone, and expression of macroscopic current through BK_Ca channels was measured as described in many previous studies, including those cited in the preceding text. We observed that overexpression of 1-GFP fusion proteins in ciliary neurons resulted in a substantial and significant increase in macroscopic Ca²⁺-dependent outward current compared with that observed in ciliary neurons transfected with GFP (Fig. 5). In experiments in which longer (450-ms) step pulses were used, it was possible to observe that expression of 1-GFP caused slowing of activation compared with Ca²⁺-dependent outward currents observed in cells expressing GFP (Fig. 5A, bottom right). Therefore the effects of the avian 1-subunit are not limited to a transformed cell line derived from nonneuronal cells, and may play a role in neuronal systems as well.

N-terminal domains of the 1-subunit are sufficient to stimulate cell surface expression of VEDEC channels

A recent study of chimeric mammalian 1- and 2-subunits showed that both the N- and C-terminal intracellular domains of these proteins contribute to changes in BK_Ca channel gating properties seen when -subunits are present (Orio et al. 2006). Moreover, the cytoplasmic C-terminal of the human 1-subunit contains a crucial di-leucine-like IL motif that functions as an endocytic signal, and mutations in that region abolish this activity (Toro et al. 2006). Interestingly, the chicken 1-subunit contains a valine residue at the corresponding location of isoleucine-186 in that motif, and this may explain why the avian subunit does not appear to inhibit surface expression of QEERL. These observations raise the issue of what portions of avian 1-subunit are involved in stimulation of cell surface VEDEC expression. To address this issue, we prepared a series of deletion constructs and point mutations to determine which regions of the avian 1 are able to bind to Slo1 -subunits and to subsequently determine which of these regulate VEDEC trafficking. A series of 1 deletion constructs and their binding to VEDEC are summarized in Figs. 6 and 7 . Binding was assessed using two different methods: in one set of experiments, we used co-immunoprecipitation to examine binding of GFP-tagged 1 deletion mutants to VEDEC (Fig. 6, A and B). HEK293T cells were transfected with full-length 1-GFP or truncated 1-GFP fusion proteins. These cells were simultaneously co-transfected with N-terminal Myc-tagged VEDEC channels. We observed that Myc-tagged VEDEC channels could be readily detected in immunoprecipitates of full-length 1-GFP isolated from cell lysates using an antibody against GFP (Fig. 6A). Co-immunoprecipitation with VEDEC also occurred in HEK293T cells expressing a truncated 1-subunit lacking the last 45 C-terminal residues (1C-GFP) and to a lesser extent with a 1-subunit lacking the first 40 N-terminal residues (1N-GFP; Fig. 6B). However, a deletion mutant that lacked both the C- and N-terminal cytoplasmic domains but that contained the two membrane-spanning helices and the extracellular loop (1L-GFP) was not able to immunoprecipitate with VEDEC. Importantly, small GFP fusion proteins composed of only portions of the N-terminal (1N-GFP) and C-terminal (1C-GFP) were able to interact with VEDEC based on co-immunoprecipitation (Fig. 6B). These deletion constructs were robustly expressed in HEK293T cells based on GFP fluorescence (Fig. 6C).

View larger version (15K): [in this window] [in a new window]   FIG. 7. Analysis of binding of 1 deletion mutants to VEDEC by GST-pulldown assay. GST-1 fusion proteins are shown in schematic (top) and their ability to pull down N-terminal Myc-tagged VEDEC is shown below. Note that the cytoplasmic N- and C-terminal portions of 1 interact with VEDEC, but this does not occur with transmembrane and extracellular domains (GST-1L).

We next examined which portions of the 1-subunit regulate trafficking of VEDEC channels. We observed that co-expression of the GFP-1N fusion protein was sufficient to stimulate movement of VEDEC channels to the plasma membrane. However, the GFP-1C and GFP-1L fusion proteins were not able to stimulate movement of VEDEC channels to the plasma membrane. These conclusions are based on indirect monitoring by whole cell recordings (Fig. 8A) and direct monitoring of VEDEC proteins using surface biotinylation assays (Fig. 8C). Indeed, we observed that a small cytoplasmic fragment composed of the first 42 residues of 1 is as effective as the full-length 1 in enhancing macroscopic current (Fig. 8A). Supplemental Fig. 2 shows that the deletion proteins yielded robust GFP expression in HEK293T cells, suggesting they were not being degraded or grossly misfolded. Finally, we observed that mutation of some of the N-terminal residues that are conserved in many BK_Ca -subunits eliminate their ability to stimulate surface expression. Thus we individually mutated K4, E14, and L20 to alanine and expressed them as full-length GFP-fusion proteins in HEK293T cells co-expressing VEDEC forms of Slo1. None of these mutant 1-subunits was able to increase macroscopic current, whereas mutation of two nearby residues (T15 and R16) to alanine resulted in proteins that were as effective as wild-type 1 (Fig. 8B). These data indicate that although the C- and N-terminal domains of the avian 1 are able to bind to Slo1, interactions with N-terminal cytoplasmic domains are both necessary and sufficient to stimulate trafficking of BK_Ca channels.

View larger version (21K): [in this window] [in a new window]   FIG. 8. Effects of 1-subunit deletion and point mutants on trafficking of VEDEC channels to the plasma membrane of HEK293T cells point to key role of N-terminal cytoplasmic domain. A: robust stimulation of mean macroscopic currents by co-expression of full-length 1-GFP or 1N-GFP with VEDEC channels in HEK293Tcells (P < 0.05). By contrast, co-expression of 1C-GFP or 1L-GFP has no effect on mean current amplitudes, which are not significantly different from those observed when VEDEC channels are expressed by themselves. B: summary of results from N-terminal point mutants (K4A, E14A, T15A, R16A, and L20A). These proteins contained C-terminal GFP tags and recordings were made from fluorescent cells. Note significant (P < 0.05) stimulation of current through VEDEC channels in cells co-expressing the T15A and R16A mutants but lack of stimulation in the other point mutants. Control cells (Con) expressed GFP along with VEDEC. C: cell-surface biotinylation assay showing much greater cell-surface expression of VEDEC channels in HEK293T cells co-transfected with 1-GFP or 1N-GFP compared with cells co-expressing 1L-GFP or 1C-GFP.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

The Slo1 gene, which encodes the principal pore-forming -subunits of BK_Ca channels, is expressed in a large number (20) of different splice variants that often have different Ca²⁺-sensitivities and gating kinetics. Two different C-terminal splice variants of Slo1 show different patterns of trafficking in HEK293T cells. Thus when expressed by themselves, the VEDEC isoforms appear to be largely retained in intracellular compartments, whereas the QEERL isoforms exhibit a much higher constitutive level of expression on the plasma membrane. A more-detailed analysis of the mechanisms that underlie the differences between constitutive trafficking of VEDEC and QEERL channels will be the subject of a subsequent report.

The central observation of the present study is that a 1-subunit, isolated in this case from chick parasympathetic neurons and earlier from avian sensory cells and fibroblasts (Oberst et al. 1997; Ramanathan et al. 1999), can stimulate steady-state expression of VEDEC channels on the plasma membrane. It has long been known that -subunits can affect the kinetics and the voltage and Ca²⁺ dependence of functional BK_Ca channels (Lu et al. 2006; Meredith et al. 2004). Indeed, the effects of the avian 1-subunit on BK_Ca gating have been described in considerable detail in previous studies in HEK293 cells (Ramanathan et al. 1999, 2000). Those studies showed that co-expression of avian 1 with any Slo1 -subunit isoform substantially slowed gating kinetics, especially deactivation kinetics, and also caused an increase in the Ca²⁺ dependence of the resulting BK_Ca channels. The effects on gating are qualitatively similar but somewhat smaller in amplitude than those produced by mammalian 1-subunits, which share only 40% sequence similarity with the avian homologue (Ramanathan et al. 2000). Our observations on BK_Ca gating kinetics in HEK293T cells, although made under different recording conditions, are consistent with the earlier detailed reports on 1-subunits.

The present data also show that whereas the N- and C-terminals of 1 are both capable of binding to -subunits, the N-terminal domain is both necessary and sufficient to stimulate trafficking of the VEDEC isoform to the plasma membrane. In this regard, Toro et al. (2006) recently showed that the human 1-subunit contains an endocytic signal in its C-terminal that results in reduction in the steady state surface expression of co-expressed QEERL-type Slo1 subunits. These workers concluded that this effect was caused at least in part by a motif on human 1 with the sequence YLSIL, which contains two putative overlapping endocytic signals (Y-X-X- and I/l-L) (Bonifacino and Traub 2003). Converting the serine and isoleucine in that sequence into alanines, thereby eliminating the di-leucine-like motif (I/l-L), abolished the endocytic activity of human 1. By contrast, the avian 1-subunit does not appear to inhibit surface expression of QEERL, possibly because the corresponding region has the sequence YVSVL, and therefore lacks the crucial di-leucine-like motif. It is possible that -subunits that lack this particular endocytic trafficking motif (such as chicken and possibly rat 1-subunits and all of the mammalian 4-subunits) will instead stimulate surface expression of some or all of the -subunits that are otherwise retained in intracellular pools owing to motifs in the cytoplasmic N-terminal of -subunits. In support of this hypothesis, we note that point mutations of several residues in the N-terminal that are conserved in known 1-subunits eliminate the ability of avian 1 to stimulate surface expression of VEDEC. VEDEC channels are not the only Slo1 splice variants that are retained in intracellular pools. There is a distinct class of Slo1 splice variants that contain 33 extra residues within the first transmembrane (Zarei et al. 2001) that leads to an ER retention signal (CVLF) in the intracellular linker between the first two transmembrane domains (Zarei et al. 2004). Those variants are retained in the ER of HEK293T cells (Zarei et al. 2001). It would be interesting to determine if any of the BK_Ca -subunits could stimulate surface expression of those Slo1 isoforms.

One question that emerges is whether regulated trafficking of BK_Ca channels is physiologically important in native cells. We have previously shown that two populations of neurons in the developing chick ciliary ganglion regulate BK_Ca trafficking in different ways. In the so-called ciliary neurons, trafficking of BK_Ca channels to the plasma membrane is dependent on growth factor-evoked activation of PI3K/Akt signaling cascades (Cameron et al. 1998, 2001; Chae et al. 2005a; Lhuillier and Dryer 2002). However, choroid neurons at the same developmental stages (between embryonic days 9 and 13) exhibit robust increases in surface BK_Ca channels without the need for growth factor treatment during that window (Cameron and Dryer 2000). Moreover, the BK_Ca channels of choroid neurons have markedly slower gating kinetics than those of ciliary neurons (Cameron and Dryer 2000), although they are not as slow as the ones that we have observed here in HEK293T cells or ciliary cells overexpressing 1. Transcripts encoding VEDEC channels and 1-subunits can be readily detected in the chick ciliary ganglion by RT-PCR at relevant developmental stages; indeed, that is the tissue source that was used to obtain the 1 clone used in the present studies. Those observations, together with the present results, are consistent with a model in which BK_Ca channel stoichiometry—including both the splice variant of the -subunit, and the nature of the -subunit(s)—determines the extent to which channel trafficking is a regulated process. Thus in the ciliary ganglion, expression of VEDEC isoforms in both populations of neurons, along with selective expression (or at least enhanced relative expression), of 1-subunits in choroid neurons, could explain some of the differences in gating and trafficking in the two cell types. Alternatively, 1-subunits could be subjected to different forms of posttranslational modification in choroid and ciliary cells that cause them to function differently with respect to regulation of channel trafficking. Both of these models are consistent with our observation that overexpression of 1-subunits markedly increases the density of macroscopic Ca²⁺-activated K⁺ current in embryonic day 9 ciliary neurons. This current is normally quite small or undetectable in ciliary neurons at that developmental stage owing to a primarily intracellular localization of BK_Ca -subunits (Chae et al. 2005a,b; Dourado and Dryer 1992; Lhuillier and Dryer 2002), which include VEDEC isoforms (Kim and Dryer, unpublished data).

In summary, we have shown that the avian 1-subunit of BK_Ca channels is able to stimulate surface expression of the VEDEC isoform of the Slo1 gene in a heterologous expression system as well as in a native population of neurons. This subunit does not produce a significant effect on the steady-state surface expression of QEERL isoforms. Finally, cytoplasmic domains on the N-terminal side of 1 are necessary and sufficient to stimulate VEDEC trafficking.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-32748.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We thank Dr. Min Li of Johns Hopkins University for providing Myc-tagged VEDEC and QEERL constructs and for helpful discussions.

	FOOTNOTES

 
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.

¹ The online version of this article contains supplemental data.

Address for reprint requests and other correspondence: S. E. Dryer, Dept. of Biology and Biochemistry, University of Houston, 4800 Calhoun, Houston, TX, 77204-5001 (E-mail: SDryer{at}uh.edu)

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