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The β1 Subunit of L-Type Voltage-Gated Ca²⁺ Channels Independently Binds to and Inhibits the Gating of Large-Conductance Ca²⁺-Activated K⁺ Channels

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

Received August 8, 2007; accepted November 7, 2007

	Abstract

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Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels encoded by the Slo1 gene are ubiquitously expressed, and they play a role in regulation of many cell types. In excitable cells, BK_Ca channels and voltage-activated Ca²⁺ channels often form functional complexes that allow the cytoplasmic domains of BK_Ca channels to lie within spatially discrete calcium microdomains. Here, we report a novel protein interaction between the β1-subunit of L-type voltage-activated calcium channels (Ca_vβ1) and critical regulatory domains of Slo1 that can occur in the absence of other proteins. This interaction was identified by a yeast two-hybrid screen, and it was confirmed by confocal microscopy in native neurons, by coimmunoprecipitation, and by direct binding assays. The Ca_vβ1 subunit binds within the calcium bowl domain of Slo1 that mediates a portion of high-affinity Ca²⁺ binding to BK_Ca channels and also to a noncanonical Src homology 3 (SH3) domain-binding motif within Slo1. Binding of Ca_vβ1 markedly slows Slo1 activation kinetics, and it causes a significant decrease in Ca²⁺ sensitivity in inside-out and in dialyzed cells, even in the absence of pore-forming subunits of voltage-gated Ca²⁺ channels. The guanylate kinase domain of Ca_vβ1 mediates Slo1 regulation through its binding to calcium bowl domains, and this domain of Ca_vβ1 is necessary and sufficient for the observed effects on BK_Ca activation. Binding of Ca_vβ1 to SH3-binding motifs may stabilize the interaction with Slo1, or it may contribute to formation of other complexes, but it does not seem to affect Ca²⁺-dependent gating of Slo1. Binding of Ca_vβ1 does not affect cell surface expression of Slo1 in human embryonic kidney 293T cells.

The pore-forming subunits of BK_Ca channels are encoded by the Slo1 gene (also known as KCNMA1), and they contain a core region with seven membrane-spanning domains. In addition, Slo1 proteins have an unusually large cytoplasmic tail that is subjected to extensive alternative splicing, yielding channel variants with markedly different voltage and Ca²⁺ sensitivities (Shipston, 2001). The allosteric relationship between membrane potential and cytosolic Ca²⁺ in the control of BK_Ca gating has been studied extensively (Magleby, 2003). Voltage regulation of channel gating is associated with domains in the core regions of BK_Ca channels (Díaz et al., 1998), whereas the regulator of conductance of K⁺ (Jiang et al., 2001; Xia et al., 2002) and calcium bowl domains (Schreiber and Salkoff, 1997; Bao et al., 2002; Zeng et al., 2005) in the cytoplasmic tail account for most of the Ca²⁺ regulation, although additional low-affinity Ca²⁺ binding sites may also modulate Ca²⁺ sensitivity (Zhang et al., 2001; Xia et al., 2002; Bao et al., 2004).

Although some BK_Ca channels can respond over a broad range of free Ca²⁺ concentrations (Thurm et al., 2005), most variants typically require micromolar concentrations for robust activation. For this reason, Slo1 channels must be close to a significant source of Ca²⁺ for the necessary Ca²⁺ concentrations to occur in space and time. In most excitable cells, the usual sources are voltage-gated Ca²⁺ (Ca_v) channels. Indirect evidence for the presence of BK_Ca channels within Ca_v-dependent "calcium microdomains" was documented several years ago (Wisgirda and Dryer 1994; Gola and Crest, 1993; Marrion and Tavalin, 1998), and these types of observations have been quantitatively refined in recent years (Prakriya and Lingle, 2000). A more recent study directly demonstrated the existence of macromolecular complexes between Slo1 and Ca_v1.2 (L-type), Ca_v2.1 (P/Q-type), and Ca_v2.2 (N-type) subunits in rat brain and in heterologous expression systems (Berkefeld et al., 2006).

In parasympathetic neurons of the chick ciliary ganglion, BK_Ca channels are functionally coupled to dihydropyridine-sensitive L-type Ca_v channels (Wisgirda and Dryer, 1994). L-type Ca²⁺ channels are made up of a single pore-forming subunit (Ca_v1.1 or Ca_v1.2), often in a complex with β-subunits (Ca_vβ), which regulate the gating properties and trafficking of the resulting channels (De Waard et al., 1994; Brice et al., 1997; Dolphin, 2003). The net effect of Ca_vβ subunits is to increase L-type current and the resulting voltage-evoked Ca²⁺ influx in cells where they are expressed. The Ca_vβ subunits are members of the membrane-associated guanylate kinase family of proteins. They have a Src homology 3 (SH3) domain linked by a flexible loop to a guanylate kinase (GK)-like domain (Chen et al., 2004; Van Petegem et al., 2004). Their interaction with Ca_v1.1 is reversible (Bichet et al., 2000), and the GK domain of Ca_vβ is free to interact with other proteins (Chen et al., 2004; Van Petegem et al., 2004; Hidalgo et al., 2006).

In the present study, we show that an L-type Ca_v channel β-subunit (Ca_vβ1), physically associates with Slo1 subunits of BK_Ca channels at two distinct regions: a noncanonical SH3 domain-binding motif, previously shown to link BK_Ca channels to the actin cytoskeleton via the adaptor protein cortactin (Tian et al., 2006), and also at sites within the calcium bowl region of Slo1. We were surprised to find that the Ca_vβ1-Slo1 interaction does not require the presence of other channel subunits or any other proteins. Moreover, the interaction is functionally significant, because it markedly slows BK_Ca activation and deactivation kinetics and it causes a significant decrease in Ca²⁺ sensitivity that can be readily detected in both inside-out excised patch and whole-cell recordings. Finally, we show that the effects of Ca_vβ1onBK_Ca gating can be attributed to the interaction with the calcium bowl region of Slo1.

	Materials and Methods

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Plasmids and Antibodies. An expression plasmid (pCMV-Myc-Slo1) encoding N-terminal (ectofacial) Myc-tagged mouse Slo1 was provided by Dr. Min Li (Johns Hopkins University, Baltimore, MD). A plasmid encoding full-length human Ca_vβ1 (accession no. NM_000723 [GenBank] .3) was obtained from Origene (Rockville, MD). Other plasmids, including pGBKT7-Slo1_G785-A985, pDsRed-Ca_vβ1, pCMV-HA-Ca_vβ1, pGEXKG-Slo1_G785-A985, pGEXKG-Slo1_G785-L843, pGEXKG-Slo1_Q844-I883, pGEXKG-Slo1_T884-N936, pGEXKG-Slo1_I937-A985, pcDNA 3.1(-)-Ca_vβ1, pcDNA 3.1(-)-Ca_vβ1_M1-D224, and pcDNA 3.1(-)-Ca_vβ1_P162-R598, were constructed in our laboratory using standard methods. The fidelity of all constructs was confirmed by sequencing. Mouse anti-Myc 9B11 and mouse anti-HA 2367 (Cell Signaling Technology Inc., Danvers, MA) were used for immunoblot analysis, whereas FITC-conjugated goat anti-Myc ab1263 (Abcam, Cambridge, MA) was used only for labeling of intracellular stores of Slo1 channels in confocal microscopy. Immunoblot and confocal analyses of endogenously expressed channel subunits were carried out with a rabbit antibody against mouse Slo1 (AB5228; Chemicon International, Temecula, CA) and a monoclonal antibody against Ca_vβ1 (H00000782-M01; Abnova, Heidelberg, Germany), as described below.

Yeast Two-Hybrid Screen. This screen was carried out using the Matchmaker system (BD Biosciences, San Jose, CA) according to the manufacturer's instructions, as described in detail previously (Kim et al., 2007a). In brief, a cDNA library of embryonic day 9 (E9) chick ciliary ganglion (CG) prepared in our laboratory was screened using a construct encoding amino acids 785 to 985 of mouse Slo1 cloned into the pGBKT7 bait vector. Colonies expressing potential interacting proteins were identified by blue-white selection carried out on a quadruple dropout medium supplemented with 5-bromo-4-chloro-3-indolyl -D-galactopyranoside. After selection, the pGADT7 plasmids encoding fragments of putative interacting proteins were isolated from yeast colonies, sequenced, and subjected to BLAST search analysis.

Coimmunoprecipitation, GST Pull-Down Assays, and in Vitro Binding Assays. Immunoblot analyses, coimmunoprecipitation, and glutathione transferase (GST) pull-down assays were carried out as described previously (Kim et al., 2007a). The in vitro binding assay is a slight modification of the GST pull-down assay. Full-length Ca_vβ1 cDNA was cloned into the pcDNA3.1(-) vector for in vitro transcription and translation using the TNT T7 quick-coupled transcription/translation system (Promega, Madison, WI). Biotinylated lysine residues were incorporated into the in vitro translated protein using Trancend tRNA (Promega), which allowed for chemiluminescent detection of Ca_vβ1 on nitrocellulose membranes using the Trancend nonradioactive detection system (Promega). Glutathione-Sepharose 4B beads carrying GST-fusion proteins prepared from Slo1 were incubated with in vitro-translated Ca_vβ1inPBS containing 0.2% Triton X-100 (PBST) overnight at 4°C with gentle rotation. The beads were washed repeatedly in PBST and eluted in a buffer containing 10 mM glutathione. Eluates were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and analyzed by addition of a streptavidin-horse-radish peroxidase conjugate (1:10,000 dilution in a Tris-buffered saline containing 0.5% Tween 20) followed by detection of labeled proteins by chemiluminescence.

Cell Culture and Transfection. Human embryonic kidney (HEK) 293T cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO₂ incubator. Transfection procedures are described in Kim et al. (2007a). Cells were used for physiology or biochemistry 48 h after transfection. Neurons from E9 chick ciliary ganglia were dissociated and cultured as described previously (Cameron et al., 1998; Lhuillier and Dryer, 2002).

Immunostaining and Confocal Microscopy. E9 CG neurons were maintained in culture for 3 or 48 h, and then they were fixed in 4% paraformaldehyde for 10 min, blocked, and permeabilized with PBS containing 0.5% Triton X-100. The preparations were then incubated with rabbit anti-Slo1 (1:500 dilution) and mouse anti-Ca_vβ1 (1:1000 dilution) overnight at 4°C. After repeated washing, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit and cyanine 3-conjugated anti-mouse secondary antibodies for 1 h at 37°C, rinsed repeatedly in PBS, and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were collected on an FV-1000 confocal microscope (Olympus, Tokyo, Japan) using a Plan Apo N60x 1.42 numerical aperture oil immersion objective. Green fluorescence was evoked using an excitation wavelength of 495 nm, and emission was monitored at 519 nm. Red fluorescence was evoked by excitation at 580 nm, and emission was monitored at 620 nm.

Immunochemical Analysis of Cell Surface Slo1 Channels in HEK293T Cells. Intact HEK293T cells expressing Myc-tagged Slo1 channels in the presence or absence of Ca_vβ1 were treated with mouse anti-Myc (9B11l Cell Signaling Technology Inc.) for 20 min in normal culture medium at 37°C to label surface Slo1 channels. Cells were then rinsed in PBS, fixed in 4% paraformaldehyde for 10 min, permeabilized with PBST, blocked, and then labeled with cyanine 3-conjugated goat anti-mouse and FITC-conjugated goat anti-Myc (ab1263) for 1 to2hat room temperature. These tagged antibodies allowed visualization of surface (red fluorescence) and intracellular (green fluorescence) Slo1 channels, respectively, by confocal microscopy. Cell surface biotinylation assays were carried out as described in detail previously (Kim et al., 2007a,b,c). In brief, intact HEK293T cells were labeled with a membrane-impermeable biotinylation reagent [sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionates] (Pierce Biotechnology, Rockford, IL). Cells were then lysed, and biotinylated proteins from the cell surface were recovered by incubating lysates with streptavidin-agarose beads. A sample of the initial lysate was reserved to measure expression of total Slo1 proteins (surface + intracellular). All of the samples were separated by SDS-PAGE and analyzed by immunoblot using an antibody against the Myc tags on the Slo1 channels.

Electrophysiology and Statistics. All physiological experiments were conducted at room temperature. Recordings were made from red fluorescent HEK293T cells coexpressing Slo1 channels with red fluorescent protein (RFP)-tagged Ca_vβ1 or with RFP. Inside-out patches were excised using standard methods. In brief, fire-polished glass micropipettes were filled with a solution containing 140 mM KCl, 1.2 mM MgCl₂, 14 mM glucose, and 10 mM HEPES, pH 7.2, and they had resistances of 2 to 5 M after filling. Test solutions bathing the cytoplasmic face of the patch membrane contained 140 mM KCl, 1.2 mM MgCl₂, 14 mM glucose, 10 mM HEPES, pH 7.2, and <1 nM free Ca²⁺ buffered with 10 mM EGTA, or 1, 5, or 10 µM free Ca²⁺ buffered with 10 mM N-hydroxy-EDTA. The free Ca²⁺ concentrations in these solutions were checked using an Orion 97-20 calcium electrode (Thermo Fisher Scientific, Waltham, MA) calibrated with commercial solution standards obtained from WPI (Sarasota, FL). Currents in each test solution were evoked by a series of eight 450-ms depolarizing steps from a holding potential of -60 mV using an Axopatch 1D amplifier (Molecular Devices, Sunnyvale, CA). Currents were digitized, and then they were analyzed off-line using pCLAMP software (Molecular Devices). Because the potassium equilibrium potential in these conditions is 0 mV, large inward tail currents occur immediately after the break of the depolarizing step pulses, and some of the step commands (e.g., to -40 mV) also yield inward currents. Methods for whole-cell recordings of Slo1 currents in transiently transfected HEK293T cells have been described previously (Kim et al., 2007a,b,c). In brief, the bathing solution contained 150 mM NaCl, 0.08 mM KCl, 0.8 mM MgCl₂, 5.4 mM CaCl₂, and 10 mM HEPES, pH 7.4. The pipette solution contained 145 mM NaCl, 2 mM KCl, 6.2 mM MgCl₂, 10 mM HEPES, pH 7.2, and 5 µM free Ca²⁺ buffered with 10 mM N-hydroxy-EDTA. Note that with these solutions, the concentrations of K⁺ inside and outside the cell are reduced to prevent whole-cell currents from saturating the patch-clamp amplifier, but the potassium equilibrium potential is physiological. Voltage activation curves in inside-out patches were generated from tail currents recorded at -60 mV by plotting the fractional activation (I_tail/_tail) against the voltage of the step command V used to evoke the currents, and then fitting the resulting curves with the Boltzmann function I_tail/_tail = [1 + exp(-(V - V) qF/RT)]^-1, where I_tail is the tail current amplitude evoked by pulse V, _tail is the maximal tail current (determined from the traces in the patch evoked by steps to +80 mV in 10 µMCa²⁺), V is the voltage of half-maximal activation, q is a slope constant, F is the Faraday constant, R is the gas constant, and T is absolute temperature. A descriptive time constant related to the kinetics of channel activation (_act) was obtained by fitting the rising phase of evoked currents with a single-exponential using the Levenberg-Marquardt algorithms implemented in Origin version 7.0 software (OriginLab Corp, Northampton, MA). We tested the hypothesis that the presence or absence of Ca_vβ1 affects the Ca²⁺ sensitivity of the dependent variables V or _act by two-way analysis of variance (ANOVA), with data from six patches in each group of cells. Other quantitative data are graphically presented as mean ± S.E.M. compiled from 10 to 25 cells in each group. Designs in which a single experimental group was compared with a single control group were analyzed using Student's unpaired t test. All statistical analyses were performed using STATISTICA software (Statsoft, Tulsa, OK), with P < 0.05 regarded as significant.

Methods for whole-cell recordings and quantification of Ca²⁺-activated K⁺ currents and voltage-activated Ca²⁺ currents from chick CG neurons have been described in detail previously (Dourado and Dryer, 1992; Cameron et al., 1998). In brief, the bath solution contained 145 mM NaCl, 5.3 mM KCl, 0.8 mM MgCl₂, 5.4 mM CaCl₂, 10 mM HEPES, and 250 nM tetrodotoxin, pH 7.4. Pipette solutions contained 120 mM KCl, 2 mM MgCl₂, 10 mM HEPES, and 10 mM EGTA, pH 7.2. For Ca²⁺-free salines, all external Ca²⁺ was replaced on an equimolar basis with MgCl₂. Currents were evoked by step depolarizations from a holding potential of -40 mV, and net Ca²⁺-dependent currents were calculated by digital subtractions (control - Ca²⁺-free) and normalized for cell surface area as described previously (Dourado and Dryer, 1992). We have previously shown that all of the macroscopic outward currents observed under these conditions are carried by BK_Ca channels (Lhuillier and Dryer, 1999; Cameron and Dryer, 2000). The much smaller voltage-activated Ca²⁺ currents were measured using similar procedures, except that the bath also contained 250 nM tetrodotoxin, 10 mM tetraethylammonium, 5 mM 4-aminopyridine, and 2 µM -conotoxin, and KCl in the pipette solutions was replaced with CsCl. In the presence of -conotoxin, essentially all of the remaining Ca²⁺ current in ciliary neurons is carried by nifedipine-sensitive L-type channels (Wisgirda and Dryer, 1994).

	Results

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BK_Ca Channels Interacted Directly with Ca_vβ1. To identify proteins that regulate BK_Ca channels, we conducted a yeast two-hybrid screen from an E9 chick CG cDNA library, using a bait from the cytoplasmic C-terminal (Gly785-Ala985) of Slo1 that is conserved in most splice variants. This bait region includes the calcium bowl (Thr911-Gln933) (Schreiber and Salkoff, 1997) and a portion of the regulator of conductance of K⁺ 2 domain (Xia et al., 2002) (Fig. 1A). Ca_vβ1 emerged repeatedly in this screen. We confirmed that Slo1 binds to Ca_vβ1 by four independent methods. First, we observed that Ca_vβ1 coimmunoprecipitates with native Slo1 channels when E9 CG extracts were probed with Slo1 antibodies (Fig. 1B). This also occurred in HEK293T cells coexpressing Myc-tagged Slo1 channels and HA-tagged Ca_vβ1 subunits (Fig. 1C). HEK293T cells do not express endogenous voltage-activated Ca²⁺ channels; therefore, this result suggests that the interaction does not require the pore-forming subunits of voltage-activated Ca²⁺ channels. In addition, we carried out pull-down experiments to confirm the interaction. To do this, we prepared a GST-fusion protein encoding the bait (GST-Slo1_G785-A985), and we incubated it with chick CG extracts. We observed that GST-Slo1_G785-A985 could precipitate Ca_vβ1 subunits from chick CG extracts that could be detected by immunoblot analysis, whereas GST was ineffective (Fig. 1D). In addition, we observed colocalization of endogenous Ca_vβ1 and Slo1 subunits in cultured E9 CG neurons by confocal microscopy. Extensive colocalization of Slo1 (green fluorescence) and Ca_vβ1 (red fluorescence) was readily detectable in merged images, and this seemed to occur in intracellular pools and on the plasma membrane (Fig. 1E, top, middle) in CG neurons processed 3 h after dissociation. Colocalization of Slo1 and Ca_vβ1 also occurred in neurites that extended from neuronal somata in cells maintained in culture for longer periods (Fig. 1E, bottom).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Direct interaction between Cavβ1 and the calcium bowl and SH3 domain-binding motifs of Slo1. A, schematic representation of Slo1. The bait fragment used in the yeast-two hybrid screen is highlighted in blue. The calcium bowl and a noncanonical SH3-binding domain are indicated in yellow and magenta, respectively. B, coimmunoprecipitation of Cavβ1 and Slo1 from chick ciliary ganglion extracts. C, coimmunoprecipitation of Cavβ1 and Slo1 from extracts of HEK293T cells expressing Myc-Slo1 and HA-Cavβ1 after transient transfection. D, GST pull-down assays carried out on chick ciliary ganglion lysates show that GST-Slo1G785-A985 binds to Cavβ1. E, colocalization of Cavβ1 and Slo1 in E9 chick ciliary ganglion neurons kept in dissociated cell culture for 3 h (top) or 2 days (middle). Regions of neurites outlined by white boxes are shown at a higher magnification on the bottom panels.

Ca_vβ1 Physically Associated with Slo1 at Two Distinct Regions. To identify specific regions of Slo1 that interact with Ca_vβ1, we made constructed smaller GST-fusion proteins made up of mutually exclusive regions within our initial Slo1 bait, shown schematically in Fig. 2A. We observed that GST-Slo1_T884-N936, which includes the calcium bowl (Thr911-Gln933), was able to precipitate Ca_vβ1 from CG extracts, whereas GST, GST-Slo1_G785-L843, GST-Slo1_Q844-I883, and GST-Slo1_I937-A985 were ineffective (Fig. 2B). Recall that the calcium bowl region of Slo1 is required for a portion of high-affinity (micromolar) Ca²⁺ binding and activation of BK_Ca channels (Bao et al., 2002; Zeng et al., 2005). We also looked for potential Ca_vβ1 binding sites outside the portions of Slo1 that we used for our bait. Because Ca_vβ1 has a functional SH3 domain (Tian et al., 2006), we prepared a GST-fusion protein that includes the noncanonical SH3-binding motifs of Slo1 (GST-Slo1_E637-D677). This fusion protein was also able to precipitate Ca_vβ1 from chick CG extracts (Fig. 2B). These interactions were observed in extracts of neuronal cells that express a full complement of voltage- and ligand-gated channels, and they cannot exclude Slo1-Ca_vβ1 interactions that occur as part of a larger complex. To address that issue, we placed human Ca_vβ1 cDNA into pcDNA3.1(-) vector, and we translated it in vitro with incorporated biotinylated lysine residues to facilitate detection. The purified product was then added to various purified GST-Slo1-fusion proteins as binary mixtures. We observed that Ca_vβ1 interacted directly with GST-Slo1_T884-N936 and GST-Slo1_E637-D677 but not with the other GST-Slo1-fusion proteins (Fig. 2C). From these results, we conclude that Slo1-Ca_vβ1 interactions do not require contributions from other proteins.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Interactions between Cavβ1 and Slo1 do not require the presence of additional proteins. A, schematic representation of GST-fusion protein constructs encoding GST-Slo1G785-A985, GST-Slo1G785-L843, GST-Slo1Q844-I883, GST-Slo1T884-N936, GST-Slo1I937-A985, and GST-Slo1E637-D677. B, GST pull-down assays show that GST-Slo1I937-A985 and GST-Slo1E637-D677 bind to Cavβ1 in chick ciliary ganglion extracts. C, full-length biotin-Cavβ1 binds directly to GST-Slo1I937-A985 and GST-Slo1E637-D677 in a binary mixture. No other proteins were present in the assay.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Cavβ1 modulates the gating properties of BKCa channels. Inside-out patch were excised from HEK293T cells expressing Slo1 in the presence or absence of Cavβ1. A, representative families of currents evoked by voltage steps (-80 to +80 mV) in inside-out patches at different bath Ca2+ concentrations, as indicated. No currents were detected in Ca2+-free bath solutions (data not shown). Note slower kinetics in presence of Cavβ1. B, rising phase of currents evoked by a voltage step to +80 mV in the presence of 10 µMCa2+. These are plotted so as to facilitate comparisons of activation kinetics. C, rising phase of these types of evoked currents were fitted with single-exponential curves, and the resulting time constants were plotted against bath Ca2+. Increasing bath Ca2+ causes channels to activate more rapidly. Two-way ANOVA of this data set reveals a significant (P < 0.0001) interaction effect, indicating that the presence of Cavβ1 affects the Ca2+ sensitivity of the resulting activation kinetics. Error bars are S.E.M. D, voltage-activation curves of Slo1 and Slo1 + Cavβ1 channels obtained at different bath Ca2+ concentrations constructed from the tail currents recorded from inside-out patches, readily seen in A. Data points (mean ± S.E.M.) are shown with superimposed fitted Boltzmann curves. Bath Ca2+ concentrations (in micromolar) are indicated next to the corresponding curves. E, plot of the V values derived from Boltzmann fits at different bath Ca2+ concentrations for Slo1 and Slo1 + Cavβ1 channels. These data were analyzed by two-way ANOVA, which revealed a significant interaction effect between the response to Ca2+ and the presence of Cavβ1 (P < 0.0057).

We observed similar effects of Ca_vβ1 on the amplitudes and kinetics of whole-cell currents recorded with pipettes containing 5 µM free Ca²⁺ from transfected HEK293T cells using methods described previously. The voltage-evoked currents recorded from cells coexpressing Slo1 and Ca_vβ1-RFP were markedly different from those observed in cells coexpressing Slo1 and RFP (Fig. 4A). In particular, currents were markedly reduced in amplitude, and they were significantly slower in Ca_vβ1-RFP-cotransfected cells (Fig. 4, B and C), much as was seen with inside-out patches exposed to the same concentration Ca²⁺.

View larger version (8K): [in this window] [in a new window]   Fig. 4. Whole-cell currents from HEK293T cells expressing Slo1 in the presence or absence of RFP-tagged Cavβ1. A, families of voltage-evoked whole-cell currents in transfected HEK293T cells coexpressing Slo1 and RFP (top) or Slo1 and Cavβ1-RFP (bottom). The recording pipettes contained 5 µM Ca2+. Note smaller and slower currents in presence of Cavβ1-RFP. B, bar graph shows mean ± S.E.M. of the maximal whole-cell currents evoked by steps to +80 mV. Cells coexpressing RFP-Cavβ1 have significantly smaller currents (P < 0.01 by Student's unpaired t test). C, effect of Cavβ1 on the rising phase of representative whole-cell currents evoked by a step to +80 mV plotted so as to facilitate comparison of activation kinetics.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Overexpression of Cavβ1 affects macroscopic Ca2+-dependent K+ currents and voltage-activated Ca2+ currents in chick CG neurons. Whole-cell currents were evoked by a series of depolarizing voltage steps in the presence and absence of external Ca2+, and net Ca2+-dependent currents were obtained by digital subtraction. A, families of Ca2+-dependent voltage-evoked K+ currents in CG neurons overexpressing RFP or Cavβ1-RFP. Note smaller and slower currents in Cavβ1-RFP-transfected neurons. B, mean ± S.E.M. of the maximal Ca2+-dependent outward currents evoked by steps to +80 mV from a holding potential of -40 mV. Current densities were significantly smaller when Cavβ1-RFP was present (P < 0.01 by student's unpaired t test). C, comparison of voltage-evoked maximal whole cell L-type calcium currents recorded from neurons expressing RFP or Cavβ1-RFP, as indicated. The recording electrodes contained 150 mM CsCl and external solutions contained 0.1 µM tetrodotoxin, 10 mM tetraethylammonium, and 2 µM -conotoxin. D, mean ± S.E.M. of the maximal L-type current densities evoked from a holding potential of -40 mV. Current densities were significantly smaller in cells overexpressing Cavβ1-RFP compared with cells overexpressing RFP (P < 0.01 by Student's unpaired t test). E, normalized current-voltage relationship of L-type Ca2+ currents in CG neurons overexpressing RFP (black) or Cavβ1-RFP (gray). Data points are mean ± S.E.M. from eight cells in each group, and the curves are spline fits with no theoretical significance.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Coexpression of Cavβ1 does not alter steady-state levels of Slo1 on the surface of HEK293T cells. A, mean maximal currents (+60 mV and 10 µM Ca2+) in inside-out patches excised from HEK293T cells coexpressing Slo1 and RFP or Slo1 and Cavβ1-RFP are not significantly different. B, confocal images of the distribution of Slo1 channels in transfected HEK293T cells obtained using different primary antibodies against ectofacial Myc tags on Slo1 applied before and after cell permeabilization. Note that Cavβ1 has no effect on surface expression of Slo1 (green fluorescence) or on Slo1 channels in intracellular pools (red fluorescence). The same laser intensities were used to obtain these images. C, cell-surface biotinylation assay using antibodies against the Myc tags on Slo1 shows no significant effect of Cavβ1 on the surface expression of Slo1.

Interactions with the GK Domain of Ca_vβ1 Were Sufficient to Modulate BK_Ca Gating. Recent structural analyses of the family of Ca_vβ subunits have demonstrated that an SH3 domain and a GK domain form a bidentate interaction with the pore-forming subunits of L-type Ca²⁺ channels, resulting in changes in their gating, trafficking and stability in the plasma membrane (Takahashi et al., 2005). The noncatalytic GK domain is a structural signature of the membrane-associated guanylate kinase family of proteins, and it is a potential protein-protein interaction motif (Chen et al., 2004; Van Petegem et al., 2004) (Fig. 7A). To identify the regions in Ca_vβ1 that interact with Slo1, we carried out in vitro binding assays in binary mixtures of GST-Slo1-fusion proteins and two different purified Ca_vβ1 fragments. The latter include Ca_vβ1_M1-D224 and Ca_vβ1_P162-R598, which comprise the SH3 and GK domains, respectively. We observed that Ca_vβ1_M1-D224 binds to GST-Slo1_E637-D677, a region within Slo1 that contains a noncanonical SH3-binding motif (Tian et al., 2006). We also observed that Ca_vβ1_P162-R598 binds to GST-Slo1_T884-N936, which includes the calcium bowl domain (Fig. 7B). To examine the functional significance of these interactions, we expressed the Ca_vβ1 fragments as RFP-fusion proteins in HEK293T cells, along with full-length Slo1. We then examined Slo1 gating in red fluorescent cells using whole-cell recordings with pipettes containing 5 µM Ca²⁺, as described above. We observed that coexpression of RFP-Ca_vβ1_P162-R598 caused slowing and inhibition of Slo1 currents similar to that observed with full-length RFP-Ca_vβ1, whereas RFP-Ca_vβ1_M1-D224 had no apparent effect on these parameters of Slo1 gating (Fig. 7, C and D). This suggests that interactions with the GK domain region of Ca_vβ1 are sufficient to modulate BK_Ca gating.

View larger version (11K): [in this window] [in a new window]   Fig. 7. The GK and SH3 domains of Cavβ1 interact with Slo1, and the GK domain is necessary and sufficient to affect Slo1 gating. A, schematic diagram of Cavβ1. The SH3 and GK domains are shown in gray and black, respectively. B, in vitro binding assay showing that the Cavβ1M1-D224 fragment, which includes the SH3 domain, binds to a noncanonical SH3-binding motif in Slo1 (Tian et al., 2006). A fragment comprised of Cavβ1P162-R598, which includes the GK domain, binds to the calcium bowl region of Slo1. C, whole-cell recordings from HEK293T cells made with recording pipettes containing 5 µM Ca2+ show that the Cavβ1P162-R598 fragment inhibits and slows macroscopic currents carried by Slo1. However, the Cavβ1M1-D224 fragment does not. Bar agraphs represent mean ± S.E.M. Inset shows representative traces from HEK293T cells coexpressing the SH3-domain fragment.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In many excitable cells, BK_Ca channels are components of a feedback system that limits the amount of Ca²⁺ influx evoked by membrane depolarization. Because of these dynamics, BK_Ca channels can contribute to spike repolarization, membrane potential oscillations, and the control of muscle contractility (Salkoff et al., 2006). Several investigators have addressed the question of how BK_Ca channels can be selectively regulated by Ca²⁺ influx through Ca_v channels given that BK_Ca channels typically require micromolar concentrations of free Ca²⁺ for robust activation at physiological membrane potentials (Grunnet and Kaufmann, 2004; Berkefeld et al., 2006; Loane et al., 2007). These studies have shown that complexes made up of the pore-forming -subunits of BK_Ca channels (Slo1) and voltage-activated Ca²⁺ channels (Ca_v1.2, Ca_v2.1, and Ca_v2.2) can be copurified from mammalian brain. Although these complexes can include auxiliary (nonpore-forming) subunits of BK_Ca channels (β2 and β4) and Ca_v channels (Ca_vβ1, Ca_vβ2, Ca_vβ3, and Ca_vβ4) (Berkefeld et al., 2006), the auxiliary subunits are not necessary for BK_Ca channels to reside within the local (20 nm) and steep Ca²⁺ concentration gradient that builds up around an open Ca_v channel pore in the presence of mobile buffers (Loane et al., 2007). Moreover, not all cell surface BK_Ca channels in neural cells occur in a complex with functional Ca_v channels (Gola and Crest, 1993; Marrion and Tavalin, 1998) and the potential consequences of formation Slo1-Ca_vβ complexes in which the pore-forming -subunits of Ca²⁺ channels are missing have not been examined previously.

In the present study, we have observed a functionally significant interaction between Slo1 (BK_Ca channel) and Ca_vβ1 subunits that can occurs in the absence of other proteins and that has a profound effect on the gating properties of BK_Ca channels. Formation of a complex with Ca_vβ1 markedly slows voltage-evoked activation of BK_Ca channels, and it reduces their apparent Ca²⁺ sensitivity, leading to net inhibition of gating at moderate concentrations of cytoplasmic Ca²⁺. It is important to note that this can occur in the absence of any other Ca_v channel subunits. In addition, we observed that coexpression of Ca_vβ1 subunits did not have a marked effect on the steady-state surface expression of Slo1 subunits as detected by electrophysiology or biochemistry in HEK293T cells.

Previous studies have shown that Ca_vβ subunits interact with a conserved domain in the pore-forming -subunits of Ca_v channels known as the -interaction domain (Chen et al., 2004). The -interaction domain interacts primarily with the GK domains of Ca_vβ subunits (Chen et al., 2004), and coexpression of a fragment containing only a portion of the GK domain is sufficient to cause shifts in the voltage dependence and kinetics of Ca_v channels (Chen et al., 2004). However, complete reconstitution of all of the modulatory activities of Ca_vβ subunits, including stimulation of trafficking, occurred only when fragments separately made up of SH3 and GK domains were simultaneously coexpressed with Ca_v -subunits in HEK293 cells (Takahashi et al., 2005). The situation with Slo1 channels is somewhat different, because both of the protein interaction domains of Ca_vβ1 can bind to Slo1 channels, although they do so at different sites in the cytoplasmic C-terminal of the latter. Moreover, expression of a fragment that contained only the GK domain was sufficient to reproduce the modulatory effects of full-length Ca_vβ1 on the Ca²⁺-dependent gating properties of BK_Ca channels. It is noteworthy that the GK domain of Ca_vβ1 interacts at a site close to or within the calcium bowl domain of Slo1, a region necessary for at least a portion of high-affinity activation of BK_Ca channels by Ca²⁺ (Bao et al., 2002; Zeng et al., 2005). The calcium bowl domain seems to be especially important in regulating the kinetics of Ca²⁺-dependent Slo1 channel activation at low (1-10 µM) Ca²⁺ concentrations (Zeng et al., 2005), and it is notable that one of the largest effects of Ca_vβ1 is to slow activation of coexpressed Slo1 channels over that same concentration range. It is possible that binding of the Ca_vβ1 GK domain to Slo1 modulates Ca²⁺ activation by simple steric hindrance within the calcium bowl, or through an allosteric mechanism that entails conformational changes in the over-all Ca²⁺ binding pocket in that part of the Slo1 channel. Expression of the SH3 domain of Ca_vβ1 by itself did not affect Ca²⁺-dependent gating of Slo1 channels, but we cannot exclude that the SH3 domain plays a role in modulating other aspects of Slo1 function, possibly by allowing formation of a larger complex. In this regard, Slo1 channels have a noncanonical SH3-binding motif that has been implicated in cytoskeletal interactions that contribute to stretch-sensitive gating (Tian et al., 2006). It is possible that Ca_vβ1 forms a bridge that allows mechanical coupling between SH3-binding motif and the calcium bowl, thereby contributing to the process of stretch-sensitive gating.

One question that emerges is whether Ca_vβ1 subunits are independent targets of physiological regulation. As already noted, the portions of Ca_vβ1 that produce functional effects upon binding to Slo1 are located within the GK domain, and the crystallographic data suggest that these sites may be occluded in those Ca_vβ1 subunits that are already bound to Ca_v -subunits (Chen et al., 2004). This suggests that under most conditions, interactions between Ca_vβ and Ca_v -subunits predominate, and this is supported by the observation that Slo1 channels expressed in the presence of a combination of Ca_vβ1 and Ca_v -subunits have fast kinetics, similar to those observed when Slo1 is expressed by itself (Berkefeld et al., 2006), an observation that we have confirmed (S. Jha and S. E. Dryer, unpublished observations). Moreover, the fact that Ca_vβ1 subunits can stimulate the surface expression of Ca_v -subunits suggests that all high threshold voltage-activated Ca²⁺ channels are assembled with a β-subunit before insertion into the plasma membrane. However, it is possibly that these dynamics can be altered by changes in the relative abundance of the various subunits, but currently, very little is known about factors that regulate Cavβ at the transcriptional or translational level. However, it is worth noting that dynamin, a protein involved in endocytotic regulation of membrane-associated proteins, can bind to the SH3 domains of Ca_vβ1 even in the absence of other subunits (Gonzalez-Gutierrez et al., 2007). In addition, several members of the Rad, Gem, and Kir family of Ras-like GTPases can also interact directly with the GK domains of Ca_vβ1 subunits, leading to down-regulation of their steady-state expression on the cell surface (Béguin et al., 2001). Thus, it is possible that the amounts of Ca_vβ1 at the cell surface are regulated independently of other subunits and that their removal from the plasma membrane exposes protein interaction motifs that allow them to form new interactions (e.g., with Slo1).

The present data argue against a role for Ca_vβ1 in regulation of the trafficking of Slo1 channels, although it should be noted that this process is sometimes cell type-dependent, and HEK293T cells may not have provided the appropriate context in which to observe such an effect. Conversely, increases in the surface expression of Ca_vβ1 could increase voltage-evoked Ca²⁺ influx by potentiation of Ca_v channels, and by independently slowing and inhibiting BK_Ca channels, thereby suppressing the main negative feedback to voltage-dependent Ca²⁺ influx. In this regard, the effect of Ca_vβ1 subunits on BK_Ca channels is not saturated under normal physiological conditions, because overexpression of Ca_vβ1 in native ciliary ganglion neurons caused slowing and reduction in mean densities of macroscopic Ca²⁺-activated K⁺ channels, along with a simultaneous increase in L-type Ca²⁺ currents. Finally, we should note that all of our experiments have been carried out on Ca_vβ1 subunits, which normally contribute to formation of L-type Ca²⁺ channels. The question arises as to whether other Ca_vβ subunits can produce a similar effect on Slo1. Although we have no data to address this issue, the GK domains of this group of proteins are very highly conserved (Dolphin, 2003); therefore, it is quite possible that this is a general property of all of them. This is notable because interactions between other Cavβ subunits and Slo1 have been described in brain (Berkefeld et al., 2006; Loane et al., 2007).

In summary, we have identified a functionally significant interaction between Ca_vβ1, an auxiliary subunit of a voltage-activated Ca²⁺ channel, and Slo1 the principle pore-forming subunit of large-conductance Ca²⁺-activated K⁺ channels. Binding of Ca_vβ1 to Slo1 can occur in the absence of any other channel subunits, and the interaction produced profound affects on the Ca²⁺-dependent gating of BK_Ca channels. This interaction may comprise part of a mechanism to modulate negative feedback control of voltage-dependent Ca²⁺ influx in excitable cells.

	Acknowledgements

 
We thank Dr. Min Li for providing Myc-tagged Slo1-expression constructs.

	Footnotes

 
This study was supported by National Institutes of Health grant NS32748. S.Z. and S.J. contributed equally to this research.

ABBREVIATIONS: BK_Ca, large-conductance Ca²⁺-activated K⁺;Ca_vβ1, β1 subunit of voltage-activated Ca²⁺; Slo1, pore-forming subunit of BK_Ca channel; Ca_v, voltage-gated Ca²⁺; SH3, Src homology 3; GK guanylate kinase; HA, hemagglutinin; FITC, fluorescein isothiocyanate; E9, embryonic day 9; CG, ciliary ganglion; GST glutathione transferase; PBST, phosphate-buffered saline containing 0.2% Triton X-100; HEK, human embryonic kidney; PBS, phosphate-buffered saline; RFP, red fluorescent protein; ANOVA, analysis of variance.

Address correspondence to: Dr. Stuart E. Dryer, Department of Biology and Biochemistry, University of Houston, 4800 Calhoun, Houston, TX 77204-5001. E-mail: sdryer{at}uh.edu

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