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Am J Physiol Cell Physiol 292: C1459-C1466, 2007. First published December 13, 2006; doi:10.1152/ajpcell.00322.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

RACK1 is a BKCa channel binding protein

Christina Kaldany Isacson,1,2 Qing Lu,2 Richard H. Karas,2 and Daniel H. Cox1,2

1Molecular Cardiology Research Institute, Tufts-New England Medical Center, and 2The Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts

Submitted 12 June 2006 ; accepted in final form 6 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The large conductance calcium-activated potassium channel, or BKCa channel, plays an important feedback role in a variety of physiological processes, including neurotransmitter release and smooth muscle contraction. Some reports have suggested that this channel forms a stable complex with regulators of its function, including several kinases and phosphatases. To further define such signaling complexes, we used the yeast two-hybrid system to screen a human aorta cDNA library for proteins that bind to the BKCa channel's intracellular, COOH-terminal "tail". One of the interactors we identified is the protein receptor for activated C kinase 1 (RACK1). RACK1 is a member of the WD40 protein family, which also includes the G protein beta-subunits. Consistent with an important role in BKCa-channel regulation, RACK1 has been shown to be a scaffolding protein that interacts with a wide variety of signaling molecules, including cSRC and PKC. We have confirmed the interaction between RACK1 and the BKCa channel biochemically with GST pull-down and coimmunoprecipitation experiments. We have observed some co-localization of RACK1 with the BKCa channel in vascular smooth muscle cells with immunocytochemical experiments, and we have found that RACK1 has effects on the BKCa channel's biophysical properties. Thus RACK1 binds to the BKCa channel and it may form part of a BKCa-channel regulatory complex in vascular smooth muscle.

calcium-activated potassium channel; protein kinase C; smooth muscle

LARGE CONDUCTANCE Ca2+-activated potassium channels (BKCa channels) are uniquely sensitive to both intracellular Ca2+ concentration and membrane voltage. They are found in many tissues, and they are particularly abundant in nerve and smooth muscle, where they provide feedback control over the Ca2+ and voltage-dependent processes of neurotransmitter release and smooth muscle contraction. In the nervous system, BKCa channels are activated by Ca2+ entering through nearby calcium channels (26, 39, 41), and their activity regulates the duration of the nerve-terminal action potential (20, 40, 47). In smooth muscle, they are activated by Ca2+ released from internal stores (6, 30). This causes the membrane potential to hyperpolarize and Ca2+ entry through voltage-gated Ca2+ channels to decrease (17, 30, 31). In both cases, BKCa channels regulate tissue function indirectly by regulating Ca2+ entry.

Given the BKCa channel's role as an important regulator of Ca2+-dependent processes, it is perhaps not surprising that the BKCa channel itself is regulated by second messengers in addition to calcium, including protein phosphatases (8, 55, 56), heterotrimeric GTP binding proteins (18, 19), and protein kinases (36–38). The BKCa channel's pore-forming {alpha}-subunit is phosphorylated by cAMP-dependent protein kinase (PKA) (50, 58), protein kinase C (PKC) (38), cGMP-dependent protein kinase (PKG) (3, 48) and cSrc (24). The channel's auxiliary beta1-subunit (primarily expressed in smooth muscle) contains a clear PKG consensus sequence and a potential PKA site (51). Whether these sites are phosphorylated in vivo, however, has yet to be determined.

In general, PKA, PKG, and cSrc activate the BKCa channel (10, 24, 28, 33, 45), although some inhibitory effects of PKA and cSrc have also been described (2, 50). PKC is inhibitory (5, 21). Whether these kinases encounter their substrate randomly or exist in a complex with the channel is not completely clear; however, the preponderance of evidence suggests that often the latter is the case. For example, cGMP and MgATP alone are sufficient to activate BKCa channels in excised membrane patches from pulmonary artery smooth muscle cells, and this activation is inhibited by the PKG inhibitor H-8 (33). In addition, reconstitution experiments with brain tissue indicate that a PKC-like activity stably associates with the BKCa channel throughout the reconstitution process (8, 38). Thus, at least in certain tissues, electrophysiological experiments suggest that PKG and PKC stably associate with the BKCa channel. Indeed, cSrc, PKG, and PKA have also been shown biochemically to associate with the BKCa channel in either native tissues or heterologous expression systems (2, 48, 49, 53, 59), as has the Ca-dependent phosphatase calcineurin (25). Little is known, however, about how these proteins are organized into a regulatory complex.

To examine the nature of the BKCa channel's regulatory complex in vascular smooth muscle, we used the "tail" portion of the COOH-terminal domain of the BKCa channel to screen a human aorta yeast two-hybrid library for proteins that bind to the BKCa channel. In this way, we identified receptor for activated C kinase 1 (RACK1) (42) as a BKCa binding protein. As its name implies, RACK1 was first identified as a PKC-targeting protein however, recent studies suggest that RACK1 acts as a general scaffolding protein. Here we report that RACK1 binds to the BKCa channel in vitro and in situ, and that when RACK1 is overexpressed in Xenopus oocytes, it alters BKCa-channel gating. These results suggest that RACK1 plays a role in the physiological regulation of the BKCa channel.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Yeast two-hybrid screen. A yeast two-hybrid screen was performed according to a standard protocol. Briefly, the COOH-terminal 470 amino acids (Bait; see Fig. 1A) of hSlo11 (the gene encoding the human isoform of the {alpha}-subunit of the BKCa channel) were fused to the COOH-terminus of the DNA binding domain of the yeast transcription factor GAL4 (residues 1–147) in the parent bait plasmid pGBKT7 (Clontech, Palo Alto, CA). The PGBKT7 vector has TRP1 as a selectable marker. The hSlo1 COOH-terminal bait was screened against a human aorta cDNA library (Clontech) subcloned just downstream of the GAL4 activating domain of pACT2 (Clontech). The bait and the library were transformed into the yeast strain AH109 (Clontech), a strain designed to reduce the incidence of false positives in which three reporters are integrated at unrelated chromosomal locations (HIS3, ADE2, and lacZ under the control of the GAL1, GAL2, and MEL1 promoters, respectively). Approximately 400,000 transformed yeast colonies were screened for hSlo1-interacting clones. The cDNA insert of each positive clone was sequenced at the Tufts University School of Medicine's sequencing facility.


Figure 1
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  		Fig. 1. A: diagram of the hSlo1 subunit, four of which form a fully functional large conductance calcium-activated potassium (BKCa) channel. Indicated are the core and tail domains, the pore helix (P), hydrophobic regions (S1–S10), the calcium bowl, and the region of the COOH-terminal "tail" used to create our bait. B: a yeast two-hybrid interaction between hSlo1 and receptor for activated C kinase-1 (RACK1). Left, hSlo1-bait and RACK1 vectors transformed together into yeast yields growth. Middle, RACK1 + empty bait vector PGBKT7 yields no growth. Right, Bait + empty library vector pACT2 yields no growth.

 
Expression and purification of GST fusion proteins. An EcoRI-SalI fragment encoding 470 amino acids from the COOH-terminus of the human BKCa channel's {alpha}-subunit was subcloned in frame behind the glutathione-S-transferase (GST) of the bacterial expression plasmid pGEX-4T1 (Pharmacia Biotech). Following transformation into the Escherichia coli strain BL21, a 25 ml LB/amp (100 µg/ml) overnight culture was used to inoculate 225 ml of LB/amp. The culture was grown for 1.5–2 h (A600 = 0.8) at 37oC and then induced with isopropylthio-beta-galactoside (IPTG) (0.1 mM) for 4 h. Cells were collected and frozen. Cells were thawed at room temperature and resuspended in 10 ml of ETN washing buffer (20 mM Tris, pH 8.0; 100 mM NaCl, 1.5 mM EDTA, 0.1% sarcosyl, and protease inhibitors). Cells were kept on ice for 15 min. Additional EDTA (final concentration of 5–6 mM) and sarcosyl (final concentration of 1.4%) were added. Cells were sonicated on ice and then spun down at 10,000 rpm for 50 min at 4oC. Supernatants were collected and poured into 50-ml tubes, then divided into two tubes. To each fraction, 10 ml of 10% Triton X-100 was added and gently mixed. One fraction was frozen at –80oC. To the other fraction, 500–700 µl of blocked glutathione agarose beads (Sigma, St. Louis, MO) were added and incubated with rocking for 2 h at 4oC. The beads were then washed 3 times with 10 ml of cold pull-down buffer (10 mM Tris·HCl, pH 7.5, 130 mM KCl, 1 mM MgCl2, 1 mM EDTA, protease inhibitor cocktail, 0.5–1.0% Nonidet P-40). Beads were stored at 4oC, until used in GST pull-down experiment, in an equal volume of cold pull-down buffer with the addition of 50 ul of 2% NaN2.

GST pull down. Approximately 300 µl of glutathione agarose beads with bound purified GST-fusion protein was incubated with ~500 µg of Cos-7 cell lysate (a source of RACK1) in cold pull-down buffer for a total volume of 1 ml. Tubes were rocked at 4oC for 2 h. The beads were then washed twice with 1 ml of pull-down buffer (centrifuged at 1,000 rpm for 2 min, and rocked for 5 min at 4oC in between washes). Beads were then loaded on a 10% SDS-PAGE gel and RACK1 binding to the BKCa channel-GST fusion protein was analyzed by Western blot with a mouse monoclonal antibody against RACK1 diluted 1:250 (Becton-Dickinson Transduction Laboratories, San Diego, CA).

Co-Immunoprecipitation. Rat pulmonary artery smooth muscle cells (PAC-1)(1) were grown in phenol red free Dulbecco's modified Eagle's medium with 10% fetal bovine serum. PAC-1 cells were plated in 100 mm dishes and grown until ~90% confluent. Two dishes of cells were used for each immunoprecipitation. The cells were rinsed twice with ice-cold phosphate-buffered saline, then scraped from the dish in TLB lysis buffer (20 mM Tris, pH 7.5, 0.137 M NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 25 mM beta-glycerol phosphate), including PMSF and protease inhibitor mixture. The cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was then incubated overnight with 5 ug of mouse IgG (nonimmune IgG), mouse monoclonal anti-RACK1 antibody (Becton Dickinson Transduction Laboratories), rabbit IgG, or rabbit polyclonal anti-BKCa channel (Alomone Labs, Jerusalem, Israel). Protein G beads (Amersham Bioscience) were then added and a further incubation carried out at 4C for 2 h. The pellets obtained after centrifugation were washed five times with wash buffer (50 mM Tris, pH 7.5, 7 mM MgCl2, 2 mM EDTA, and 1 mM PMSF). The immunopellets were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-RACK1 antibody, or anti-BKCa channel. The BKCa channel protein band was confirmed by immunoblotting with a second anti-BKCa channel antibody (US Biological, Swampscott, MA) (data not shown). The presence of PKC was tested using an anti-pan PKC antibody (MC5, Santa Cruz Biotechnology).

Immunocytochemistry. Smooth muscle cells were dissociated from rat basilar arteries essentially as previously described (35). Briefly, the basilar artery of an adult rat was removed and incubated for 20 min at 37°C in ES buffer composed of (in mM) 80 Na glutamate, 5.6 KCl, 55 NaCl, 2 MgCl2, 10 HEPES, and 10 glucose, with 0.3 mg/ml papain (Worthington) and 1 mg/ml dithiothreitol added. The artery was then spun down in a benchtop centrifuge (1.5 min, 2,000 rpm) and incubated in ES buffer containing 100 µM CaCl2 and 1 mg/ml collagenase (70% Type F, 30% type H, Sigma-Aldrich, St. Louis, MO). The artery was spun down again, placed in fresh ES buffer and incubated on ice for 15 min. The smooth muscle cells were then dispersed by tritration with a pasteur pipette that had been fire polished to a diameter 1/3 its normal size, ~30 strokes. These cells were then pipetted onto laminin-coated coverslips (Becton Dickinson, Bedford, MA) and allowed to stick for 1.0–1.5 h at 37oC. Coverslips were washed with PBS, and then cells were fixed in 4% paraformaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 for 1 min. Following 1 h of blocking with 10% horse serum in PBS, cells were incubated in primary antibody for 1 h at room temperature (mouse anti-RACK1; Becton Dickinson Transduction Laboratories, San Diego, CA) at 1:50, mouse anti-BK (Alomone, Israel) at 1:200. Following a wash with PBS, cells were then incubated in secondary antibody in the dark for 1 h at room temperature: Cy3 anti Ms IgM (Jackson Immunoresearch, West Grove, PA) was used for RACK1 visualization at 1:2,000, and Alexa 488 anti-Rabbit (Molecular Probes, Eugene, OR) was used for BK visualization at 1:1,000. Cells were then washed a final time with PBS, and mounted on slides. Cells were examined using a Leica laser scanning confocal microscope at the Tufts/NEMC Imaging Facility (Boston, MA).

Electrophysiology. Electrophysiological experiments were done with the hSlo1 clone essentially as previously described (4).2 The mouse beta-subunit clone was coexpressed with hSlo1 in some of the experiments.3In vitro transcription was performed with the "mMessage mMachine" kit with T3 RNA polymerase (Ambion, Austin, TX). To record macroscopic currents ~0.5 to 50 ng of cRNA were injected into Xenopus laevis oocytes (stages IV and V) 2–6 days before recording. hSlo1 and beta-cRNA were injected in a 1:1 molar ratio. RACK1 cRNA was injected in a 1:1 ratio with the hSlo1 cRNA.

All recordings were done in the inside-out patch-clamp configuration (14) using pipettes made of borosilicate glass (VWR micropipettes, West Chester, PA), with resistances of 1–2 M{Omega} in our recording solutions. Pipette tips were coated with sticky wax (Sticky Wax, Emeryville, CA) and fire polished before use. Data were acquired using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA), "Pulse" acquisition software (HEKA Electronik, Lambrecht, Germany), and an ITC-16 AD/DA interface (Instrutech Scientific Instruments, Great Neck, NY). Currents were digitized at 50 KHz and low pass filtered at 10 KHz. All experiments were carried out at room temperature, 22–24°C. Before currents were analyzed, capacity and leak currents were subtracted using a P/5 leak subtraction protocol with a holding potential of –120 mV and voltage steps opposite in polarity to those in the experimental protocol.

Two recording solutions were used. The pipette solution was composed of (in mM) 80 KMeSO3, 60 N-methyl-glucamine-MeSO3, 20 HEPES, 2 KCl, 2 MgCl2 (pH 7.20). The internal solution was composed of (in mM) 80 KMeSO3, 60 N-methyl-glucamine-MeSO3, 20 HEPES, 2 KCl, 1 HEDTA, and CaCl2 sufficient to give 10 µM free Ca2+ concentration (pH 7.20). The appropriate amount of total Ca2+ (100 mM CaCl2 standard solution, Orion Research, Boston, MA) to add to the base internal solution to yield the desired 10 µM free Ca2+ concentration was calculated using the program Max Chelator (7), which was downloaded from (www.stanford.edu/~cpatton/maxc.html), and the proton and Ca2+-binding constants of Bers (supplied with the program) for pH 7.20, T = 23°C, and an ionic strength of 0.15. Free solution was then measured with a Ca2+-sensitive electrode (Orion Research).

Conductance-voltage (G-V) relations were determined from the amplitude of tail currents measured 200 µs after repolarization to a fixed membrane potential (–80 mV) after voltage steps to the indicated test voltages. Each G-V relation was fitted with a Boltzmann function: G = Gmax/[1 + e – zF(V – V1/2)/RT], and normalized to the peak of the fit. All curve fitting was done with "Igor Pro" graphing and curve fitting software (WaveMetrics, Lake Oswego, OR) using the Levenberg-Marquardt algorithm to perform nonlinear, least squares fits. This software was also used to fit an exponential function to the time course of BKCa current activation.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of RACK1 as a possible BKCa-channel-interacting protein. To identify proteins that interact with the BKCa channel, a sequence encoding 470 amino acids of the COOH-terminal "tail" of hSlo1 (the human isoform of the BKCa channel's {alpha}-subunit) was used as bait in a yeast two-hybrid screen (13) of a human aorta smooth muscle cDNA library (Fig. 1A). Several tentative positives were identified, and of these, we pursued most vigorously RACK1, Receptor for Activated C Kinase 1 (MW 36 KDa). As its name implies RACK1 was originally described as a targeting protein for activated PKCbetaII (12, 13, 28, 42, 46); however, since then it has been found to bind to a wide range of other proteins (27). To confirm RACK1's interaction with the tail region of hSlo1 in yeast, the RACK1 clone was retransformed into yeast with the hSlo1 bait, and again the RACK1-hSlo1 interaction was observed (Fig. 1B, left). In addition, the empty bait plasmid (PGBKT7) did not show an interaction with the RACK1 clone (Fig. 1B, middle), nor did the empty library plasmid (PACT2) interact with the hSlo1 bait (Fig. 1B, right). Thus RACK1 and hSlo1 interact specifically in yeast.

hSlo1 and RACK1 interact in vitro. To examine biochemically the interaction we observed in yeast, we performed GST pull-down experiments. The tail of hSlo1 was expressed in Escherichia coli as a GST-fusion protein, BK-GST, and purified on glutathione-agarose beads. Cos-7-cell lysate, a source of RACK1, was then passed over the BK-GST-bound beads, and after a 2-h incubation and two washes, proteins associated with the beads were separated by SDS-PAGE, transferred to PVDF membranes, and probed with an anti-RACK1 monoclonal antibody. As shown in Fig. 2, in this assay, RACK1 associates with the tail of the hSlo1 channel, BK-GST (lane 6), but it does not bind to the beads alone (lane 5), or to beads attached to GST alone (lane 4). Thus, as in yeast, in vitro RACK1 specifically associates with the tail region of the BKCa channel. We also probed the BK-GST precipitate for the presence of conventional PKCs, but none were found (data not shown).


Figure 2
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  		Fig. 2. RACK1 binds to the BKCa channel in pull-down assays. Lane 6, RACK1 from Cos-7 cells binds to the BK-glutathione-S-transferase (GST) fusion protein immobilized on glutathione-conjugated agarose beads. Lane 5, no such interaction is observed when beads without the fusion protein are used. Lane 4, no such interaction is observed when GST alone is immobilized on the beads. Lanes 1–3, samples of the final wash solution for each condition (lanes 4–6) demonstrating that no residual RACK1 remained on the beads due to poor washing.

 
The BKCa channel and RACK1 interact in situ. To determine whether the BKCa channel interacts with RACK1 in smooth muscle, we immunoprecipitated the BKCa channel from PAC-1 cells (a rat pulmonary artery smooth muscle cell line that we found expresses both RACK1 and the BKCa) and tested for the presence of RACK1 by Western blot analysis. As shown in Fig. 3, when a BKCa-channel antibody was used for the immunoprecipitaion, RACK1 did coimmunoprecipitate with the channel (Fig. 3, top, lane 2), and when the reverse experiment was performed, the BKCa channel co-immunoprecipitated with RACK1 (Fig. 3, bottom, lane 3). Thus, as in yeast and in vitro, RACK1 associates with the BKCa channel in smooth muscle cells.


Figure 3
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  		Fig. 3. RACK1 coimmunoprecipitates with the BKCa channel and vice versa. Immunoprecipitations were performed with PAC1-cell lystates, and the precipitates were then blotted with either an anti-RACK1 (top) or an anti-BKCa (bottom) antibody. The precipitating antibodies were as follows: lane 1, nonimmune IgG; lane 2, anti-BKCa; and lane 3, anti-RACK1.

 
It is interesting to note the difference in the intensity of the bands of RACK1 precipitated from the cell lysates (top, lane 3) vs. RACK1 co-immunoprecipitating with the BKCa channel (top, lane 2). It appears that only a fraction of the RACK1 in the cells associates stably with the BKCa channel, a finding that is not surprising given that recent reports implicate RACK1 as a general scaffolding protein that may participate in several different pathways. Also, by comparing lanes 2 and 3 on the bottom, it appears that only a small percentage of the total BKCa channel protein is associated with RACK1, but it could also be that the RACK1-BKCa-channel interaction is weak and in part lost during the precipitation process.

To determine whether PKC is part of the BKCa-RACK1 complex, we probed for PKC in our anti-BKCa precipitates by western blot with the anti-pan ({alpha}-, beta-, {gamma}-PKC) antibody. Because RACK1 binds to the activated form of PKC, before homogenization we stimulated the PAC-1 cells for 10 min with 1 µM phorbol 12-myristate 13-acetate (PMA), an activator of PKC; however, we did not observe PKC co-precipitating with the channel (data not shown).

Partial co-localization of BKCa channels and RACK1 in vascular smooth muscle. To study the distribution of RACK1 and the BKCa channel in intact cells, freshly dissociated smooth muscle cells from the rat basilar artery were isolated and plated on laminin-coated coverslips. These cells have been shown previously to have large BKCa currents (35), and indeed, as shown in green in Fig. 4 (all panels), when these cells were stained with an anti-hSlo1 antibody, BKCa channels could be seen to outline the plasma membrane. Some internal staining was also present; this most likely represents BKCa channels that have yet to move from the golgi to the plasma membrane. More interesting, however, when these cells were stained for RACK1, RACK1 was also observed at or near the plasma membrane (shown in red in Fig. 4). In some places (arrows, Fig. 4), RACK1 and the BKCa channel appear to be co-localized. At many other places, however, the staining for the two proteins does not overlap, such that it appears that some RACK1 is localized near BKCa channels, but many BKCa channels either do not have an associated RACK1, or this association is not well visualized. This is consistent with our coimmunoprecipitation results.


Figure 4
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  		Fig. 4. Immunocytochemical results from freshly dissociated rat basilar artery smooth muscle cells. Three typical cells are shown as viewed under the laser scanning confocal microscope. BKCa channel immunostaining is shown in green. RACK1 immunostaining is shown in red. Some regions of co-localization are indicated with arrowheads.

 
In Chinese hamster ovary cells RACK1 has been found predominately in the cytoplasm around the nucleus and not at the plasma membrane (43). Thus specific mechanisms may exist in smooth muscle for the translocation of RACK1 to membrane or submembrane sites. We did not observe any change in the distributions of RACK1 or hSlo1 when the smooth muscle cells were pretreated with the PKC activator, PMA (100 nM) (data not shown), but it could be that some other means of activation is required to translocate RACK1 to the BKCa channel.

Effects of RACK1 overexpression on BKCa channel currents in Xenopus oocytes. To determine whether RACK1 influences the biophysical properties of the BKCa channel, hSlo1, with or without the BKCa beta1 subunit, and RACK1 (human) were coexpressed in Xenopus oocytes, and macroscopic BKCa currents were recorded in the inside-out patch-clamp configuration. When the {alpha} subunit was expressed with RACK1, the channel's G-V relation with 10 µM internal [Ca2+] was right-shifted from a half-maximal activation voltage of 59 ± 3.5 (SE) mV to 72 ± 4.4 (SE) mV (P = 0.037) (Fig. 5C). 10 µM [Ca2+] was used in this experiment because that is the Ca2+ concentration BKCa channels are thought to be exposed to in smooth muscle during their natural stimulus, the Ca2+ spark (34, 35, 60). Surprisingly, however, when the same experiment was performed with the BKCa beta1 subunit present, RACK1 no longer shifted the channel's G-V relation (Fig. 5E), but it did decrease the {alpha}-beta1 channel's activation time constant (Fig. 5, B and F), an effect that was not evident in the absence of beta1 (Fig. 5D). Thus RACK1 affects the BKCa channel's biophysical properties both with and without beta coexpression. Its effects are primarily on channel kinetics when beta1 is present, as would be the case in smooth muscle, and they are primarily on the equilibrium properties of gating, when beta is absent.


Figure 5
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  		Fig. 5. Effects of RACK1 on the BKCa channel's biophysical properties. A: Western blot demonstrating overexpression of RACK1 in Xenopus oocytes. Lane 1, the endogenous level of RACK1 protein in Xenopus oocytes. Lane 2, RACK1 protein level after RACK1 and hSlo1 cRNA injection. B: current traces from {alpha} + beta channels recorded with voltages steps from –80 to +100 mV, with 10 µM [Ca2+] in the absence and presence of RACK1. Each trace has been normalized to its maximum. Note the faster activation of the current in the presence of RACK1. C: conductance-voltage relationship of the hSlo1 channel in the absence and presence of overexpressed RACK1 at 10 µM [Ca2+]. D: conductance-voltage relationship of hSlo1 with its betasubunit in the absence and presence of overexpressed RACK1 at 10 µM [Ca2+]. E: activation time constant as a function of voltage for the hSlo1 channel in the absence and presence of overexpressed RACK1 at 10 µM [Ca2+]. F: activation time constant as a function of voltage for hSlo1 with its beta-subunit in the absence and presence of overexpressed RACK1 at 10 µM [Ca2+]. Symbols are as indicated on the figure. Error bars represent standard error of the mean.

 
To confirm that the RACK1 cRNA that we injected into the Xenopus oocytes was being translated to protein, we performed western blots on the oocyte proteins before or after cRNA injection. As shown in Fig. 5A, the injected RACK1 cRNA was indeed expressed at a high level in the oocytes (lane 1); however, we also found that the oocytes have a significant level of endogenous RACK1 (lane 2), which might bind to the channel in our control RACK1-uninjected condition. Thus the effects of RACK1 on the BKCa channel's biophysical properties may be larger than those we have observed.

In two patches, we also examined the effect of 2 µM PMA on oocytes overexpressing BKCa {alpha}-beta and RACK1, but we found no clear effect of PMA as compare to the non-PMA-treated controls (n = 3). Thus stimulating PKC does not appear to greatly enhance RACK1's biophysical effects.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Enzymes that modify proteins often exist in complexes with their substrates, an arrangement that lends speed and specificity to signaling cascades. To bring the constituents of the complex together, proteins known as scaffolding proteins are commonly required. Here we have demonstrated that one such scaffolding protein RACK1 associates with the BKCa channel.

RACK1 was initially identified as a targeting protein for protein kinase C betaII (PKC), but it has since been found to bind to wide variety of other proteins. It has a molecular mass of 36,000 Da and it belongs to the WD40 protein family. These proteins contain varying numbers of a 40-amino-acid repeats termed a WD40 repeat (29). RACK1 is composed of 7 WD40 repeats, which puts it in the same subfamily as the G protein beta-subunit of transducin (Gbeta), a protein for which the crystal structure is known (44). The WD40 repeats of Gbeta, and therefore most likely of RACK1, form a rigid seven-blade beta-propeller structure (Fig. 6B; adapted from Ref. 27) that allows it to bind multiple proteins simultaneously. Indeed, in the interferon system RACK1 forms the backbone of a multiprotein complex that includes the IFN receptor, STAT1, Janus kinase 1, and tyrosine kinase 2 (52). A list of proteins that have been found to associate with RACK1 would contain at least 25 members (32) including two other channels: the NMDA receptor (57) and the GABAA receptor (9). Also, the tyrosine kinase cSrc (14), which, like PKC, has been shown to associate with and phosphorylate the BKCa channel (2, 24) has also been shown to bind to RACK1. Thus, given our failure to observe PKC associating with the BKCa channel via co-immunoprecipitation, perhaps in the cells we have studied RACK1 binds to the BKCa channel for a reason other than PKC targeting. Indeed, in the brain RACK1 has been found to target the tyrosine kinase Fyn to the NR2B subunit of the NMDA receptor. Interestingly, however, rather than enhancing NR2B phosphorylation by Fyn, RACK1 inhibits this reaction. Also, both RACK1 and the BKCa channel have been found to interact with the synaptic protein syntaxin 1A (22, 23) Thus RACK1 may play a role in the positioning or the regulation of the BKCa channel at the synapse.


Figure 6
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  		Fig. 6. RACK1 sequence and structure. A: amino acid sequence of the RACK1 protein. RACK1, a WD40 protein family member, has 7 WD40 repeats. Each repeat is represented in a unique color. B: beta-propeller structure of the beta-subunit of bovine transducin (44) color coded to indicate the corresponding WD repeats in RACK1 (adapted from Ref. 27). C: diagram of the RACK1 sequence. The top line depicts the entire RACK1 sequence with all 7 WD40 repeats. The bottom line represents the part of the RACK1 sequence pulled out of the library in our yeast two-hybrid screen.

 
In addition to its potential role as a targeting protein, however, we have found that RACK1 has effects on the gating of the BKCa channel. Most relevant to smooth muscle physiology, when beta is present, RACK1 slows channel activation in response to voltage steps at constant Ca2+. Furthermore, biophysical studies indicate that Ca2+ binding is not a rate-limiting step in BKCa-channel gating (11, 15), and thus, if activation in response to voltage steps is slowed, so too (it is predicated) will be activation in response to Ca2+ steps. This is relevant here because in smooth muscle, BKCa channels open in response to Ca2+ sparks: brief, spontaneous transient Ca2+ release events from the sarcoplasmic reticulum. These sparks activate nearby BKCa channels on the plasma membrane, creating outward BKCa currents known as spontaneous transient outward currents (30, 32, 60). The frequency and amplitude of spontaneous transient outward currents then influence membrane potential and indirectly smooth muscle tone (16, 32, 54). As sparks are brief events, having a typical decay time constant of ~32 ms (34), the speed with which a BKCa channel responds to Ca2+ from a spark may be as important as its Ca2+ sensitivity in determining to what degree the channel is activated by the spark, which in turn determines the influence the spark has on smooth muscle tone. Thus RACK1's biophysical effects on the BKCa channel could be physiologically relevant. Experiments with RACK1-knockout mice, however, which have yet to be generated, will be required to determine what influence RACK1's association with the BKCa channel has on the channel's activity in vivo.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by National Heart, Lung, and Blood Institute Grants P01-HL-077378 and R01-HL-64831.


   ACKNOWLEDGMENTS
 
We thank Anne Rapin for expert technical and electrophysiological assistance, Dr. Kathleen Dunlap for her helpful comments on the manuscript, and Drs. Andrew Braun and Howard Surks for technical advice and helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. H. Cox, New England Medical Center Hospitals, 750 Washington St., Box 786, Boston, MA 02111 (e-mail: dan.cox{at}tufts.edu)

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 nucleotide sequence for hSlo1 is accessible through the GenBank database under GenBank Accession Number U11058. Back

2 The nucleotide sequence for hSlo1 is accessible through the GenBank database under GenBank Accession Number U23767. Back

3 The nucleotide sequence for the mouse beta-subunit is accessible through the GenBank database under GenBank Accession Number AAD11857. Back


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