Assembly of a Ca2+-dependent BK channel signaling complex by binding to β2 adrenergic receptor
2004; Springer Nature; Volume: 23; Issue: 11 Linguagem: Inglês
10.1038/sj.emboj.7600228
ISSN1460-2075
AutoresAo Liu, Jingyi Shi, Lin Yang, Luxiang Cao, Soo Mi Park, Jianmin Cui, Steven O. Marx,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoArticle13 May 2004free access Assembly of a Ca2+-dependent BK channel signaling complex by binding to β2 adrenergic receptor Guoxia Liu Guoxia Liu Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Jingyi Shi Jingyi Shi Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Lin Yang Lin Yang Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Luxiang Cao Luxiang Cao Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY, USA Search for more papers by this author Soo Mi Park Soo Mi Park Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Jianmin Cui Jianmin Cui Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Steven O Marx Corresponding Author Steven O Marx Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Guoxia Liu Guoxia Liu Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Jingyi Shi Jingyi Shi Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Lin Yang Lin Yang Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Luxiang Cao Luxiang Cao Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY, USA Search for more papers by this author Soo Mi Park Soo Mi Park Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Jianmin Cui Jianmin Cui Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Steven O Marx Corresponding Author Steven O Marx Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA Search for more papers by this author Author Information Guoxia Liu1, Jingyi Shi2, Lin Yang1, Luxiang Cao3, Soo Mi Park1, Jianmin Cui2 and Steven O Marx 1 1Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY, USA 2Cardiac Bioelectricity Research and Training Center and Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH, USA 3Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY, USA *Corresponding author. Division of Cardiology and Center for Molecular Cardiology, Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Tel.: +1 212 305 0271; Fax: +1 212 342 0475; E-mail: [email protected] The EMBO Journal (2004)23:2196-2205https://doi.org/10.1038/sj.emboj.7600228 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Large-conductance voltage and Ca2+-activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of β-adrenergic receptors and protein kinase A (PKAc) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the β2-adrenergic receptor (β2AR) with the pore forming α subunit of BKCa and an A-kinase-anchoring protein (AKAP79/150) for β2 agonist regulation. β2AR can simultaneously interact with both BKCa and L-type Ca2+ channels (Cav1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca2+- and phosphorylation-dependent modulation of BKCa current. Our findings reveal a novel function for G protein-coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate β-adrenergic regulation of membrane excitability. Introduction Large-conductance voltage and Ca2+-activated potassium channels (BKCa), encoded by the gene Slo1 (Butler et al, 1993), are regulated extensively by alternative splicing (Lagrutta et al, 1994), phosphorylation/dephosphorylation (Chung et al, 1991) and associated regulatory proteins such as β subunits (Brenner et al, 2000). BKCa/Slo channels are activated by depolarization and elevated cytosolic Ca2+. In neurons, BKCa channels have been localized to cell bodies and nerve terminals (Knaus et al, 1996) and can functionally colocalize with Ca2+ channels at presynaptic terminals (Robitaille et al, 1993). In neurons, the channels underlie the fast after-hyperpolarization that contributes to resetting the membrane potential during an action potential (Storm, 1987). In presynaptic terminals, the channels are believed to influence synaptic transmission by hyperpolarizing the plasma membrane, thereby limiting Ca2+ influx (Storm, 1987; Lancaster et al, 1991; Robitaille et al, 1993; Joiner et al, 1998). In smooth muscle, BKCa channels hyperpolarize the membrane, thereby indirectly reducing contractility (Nelson et al, 1995). The direct regulation of BKCa mediates, in part, the bronchorelaxant and vasorelaxant properties of β agonists (Schubert and Nelson, 2001; Pelaia et al, 2002). An emerging concept in ion channel regulation is that modulation by phosphorylation is controlled by local signaling mechanisms (Marx et al, 2000, 2001; Davare et al, 2001). BKCa channels are potently modulated by reversible protein phosphorylation (Chung et al, 1991; Reinhart et al, 1991; Schubert et al, 1999; Schubert and Nelson, 2001; Zhou et al, 2001). Prior studies have established that BKCa is a substrate of protein kinase A (PKAc) (Sadoshima et al, 1988; Kume et al, 1989; Nara et al, 1998) that can activate or inhibit channel activity, depending on the splice isoform (Carl et al, 1991; Tian et al, 2001; Fury et al, 2002). BKCa channels are also phosphorylated by protein kinase C (PKC) (Minami et al, 1993; Zhou et al, 2001) and protein kinase G (PKG) (Kume et al, 1992; Alioua et al, 1998) at distinct sites (Zhou et al, 2001). To add to the complexity of regulation, cross-activation of PKG by cAMP-dependent vasodilators has been described (White et al, 2000; Barman et al, 2003). Although several studies have suggested that kinase(s) is/are tethered to the BKCa channel (Chung et al, 1991; Wang et al, 1999; Tian et al, 2003), the macromolecular complex that facilitates β-adrenergic receptor (β-AR) signaling to the BKCa channel has not been clearly elucidated. We identified the requirement of an association between the β2-adrenergic receptor (β2AR), A-kinase-anchoring protein (AKAP79/150) and BKCa channel to enhance β2 agonist regulation of the channel. Moreover, as β2ARs can dimerize (Angers et al, 2000) and simultaneously interact with both BKCa and L-type Ca2+ channels (LTCC) (Davare et al, 2001), a functional macromolecular signaling complex is created that permits the rapid response to β2 agonist. Results PKAc interacts with BKCa channel BKCa channels, immunoprecipitated from brain extract, were phosphorylated in vitro by PKAc, which was inhibited by the addition of a PKA inhibitor, PKI5–24 (Figure 1A). BKCa immunoprecipitated from brain is also phosphorylated in the presence of cAMP, in the absence of exogenous PKAc. The phosphorylation induced by cAMP is inhibited by PKI, suggesting that BKCa is closely associated in vivo with an endogenous PKAc (Figure 1B) (Chung et al, 1991; Esguerra et al, 1994). This endogenous kinase bound to the channel is inactive, as the BKCa channel immunoprecipitated by brain was not phosphorylated with the exclusion of cAMP (Figure 1A). PKAc can be completely dissociated from the BKCa complex by cAMP (Figure 1C), indicating that the catalytic subunit engages the complex through a regulatory subunit (holoenzyme), rather than via a distinct site on the catalytic subunit, as is the case for PKAc and Drosophila BKCa (dSlo) interaction (Zhou et al, 2002). When cAMP was not preincubated with the immunoprecipitates prior to in vitro phosphorylation, a strong phosphorylation signal is detected (Figure 1C). These data suggest that BKCa is part of a macromolecular complex that underlies the regulation of the channel by β2AR signaling/processes that elevate intracellular cAMP. Figure 1.BKCa channel is phosphorylated by associated PKA. (A) Autoradiograph (left) of BKCa channel immunoprecipitated from brain incubated with the catalytic subunit of PKA (PKAc) and [γ-32P]ATP and size-fractionated on SDS–8% PAGE; the specificity was established using preimmune serum and PKA inhibitor, PKI5–24. Immunoblot (right) of immunoprecipitation (IP) of BKCa channel from brain extract size-fractionated on SDS–10% PAGE, demonstrating specificity of antibody. ‘cont’ represents 5% of input, ‘HC’ represents the heavy chain of IgG BKCa channel is specifically phosphorylated by exogenous PKA. (B) Autoradiograph of BKCa immunoprecipitated from brain incubated with PKAc or cAMP±PKI. cAMP activates an associated, endogenous kinase that is inhibited by PKI, indicating that PKAc is associated with the channel complex. (C) Autoradiograph of BKCa immunoprecipitated from brain, initially preincubated with 5 μM cAMP (without Mg-ATP, which is not permissive for phosphorylation of the channel), followed by in vitro phosphorylation initiated by cAMP and [γ-32P]ATP/Mg-ATP. Preincubation with cAMP releases the associated PKAc from the BKCa macromolecular complex, as indicated by the reduction of cAMP-induced phosphorylation in the pretreated lane (+). Download figure Download PowerPoint β2AR associates with neuronal and smooth muscle BKCa channel Given the importance of adrenergic input for regulating membrane excitability and BKCa function, we sought to determine whether BKCa associates with β-ARs. Although both β1AR and β2AR are expressed in the brain (Figure 2A), β2AR, but not β1AR (data not shown), is associated with BKCa (Figure 2B). BKCa colocalized/distributed with β2AR in the soma of mouse brain cortical neurons (Figure 2C) and in cerebellar Purkinje cells, and in basket and stellate cells in the molecular layer of the cerebellum (data not shown). Figure 2.β2AR is associated with BKCa channel in brain and smooth muscle. (A) β2AR and β1AR immunoblot of brain and VSMC lysates. (B) BKCa immunoblot of β2AR and preimmune immunoprecipitations from rat brain. BKCa specifically associates with β2AR. (C) Representative confocal images of immunostaining of mouse cerebral cortex for β2AR (red) and BKCa (green). BKCa and β2AR are colocalized/distributed (merged image) on the soma of the cortical neuron. Scale bar, 5 μm. (D) BKCa immunoblots of β2AR immunoprecipitations from extracts of rat bladder and aorta, human lung (lymphangiomyomatosis) and VSMCs. Immunoprecipitation specificity was demonstrated using β1AR and β2AR antibody without lysate (in VSMC samples), preimmune serum or β2AR peptide-blocked antibody (+pep) in lung and VSMC samples. β2AR specifically co-immunoprecipitates with BKCa channels. ‘cont’ is 5% input. (E) Representative confocal images of immunostaining for β2AR (green) and BKCa (red) in isolated human VSMCs. BKCa and β2AR are colocalized/distributed (merged images). Scale bar, 10 μm. Download figure Download PowerPoint BKCa channels play a significant role in the regulation of vascular (Brenner et al, 2000), pulmonary (Pelaia et al, 2002) and uterine (Wallner et al, 1995) smooth muscle contraction, and modulation of BKCa by β-ARs represents an important therapeutic target. Immunoprecipitation of β2AR from extracts of tissues enriched in smooth muscle such as bladder, aorta and lung (Figure 2D) demonstrated a specific association with BKCa channels. Negative controls included β1AR and β2AR antibody alone (without lysate), preimmune serum (with lysate) and peptide-blocked β2AR antibody (with lysate). As the regulation of arterial tone is dependent on vascular smooth muscle cell (VSMC) contractility, we sought to determine whether the complex was present in isolated VSMCs. Consistent with this role, the association of β2AR and BKCa was present in VSMC as demonstrated by co-immunoprecipitation (Figure 2D) and confocal immunofluorescence microscopy (Figure 2E). The interaction was specific, as the β1AR, although expressed in vascular smooth muscle and lung as determined by immunoblot (Figure 2A and data not shown), was not associated with the channel (Figure 2D). Formation of BKCa–β2AR–AKAP79/150 complex To examine the binding determinants for BKCa on β2AR, glutathione-S-transferase (GST) fusion proteins of the β2AR intracellular loops were prepared and assayed for their ability to interact with BKCa in brain extracts. Mapping of the interaction sites revealed that only the third intracellular (i3) loop associated with the channel (Figure 3). GST fusion proteins containing the other intracellular domains (i1, i2 and C-terminus) failed to co-precipitate the channel. Figure 3.Specific interaction of the β2AR third intracellular loop and BKCa channel. Representation (left) of β2AR; GST fusion proteins were prepared for the three intracellular loops (i1–i3) and the C-terminus (Cterm). Rat brain extracts were incubated with the GST fusion proteins bound to glutathione sepharose. For GST-i3, pull-downs were performed with increasing amounts of GST fusion proteins to demonstrate specificity of interaction. After extensive washing, samples were size-fractionated on SDS–PAGE and transferred to nitrocellulose. Immunoblots (right; upper GST antibody, lower BKCa antibody) demonstrate that only GST-i3 specifically co-precipitated BKCa channel. Download figure Download PowerPoint Prior studies have demonstrated that AKAP79/150 (Fraser et al, 2000) and gravin bind to β2AR (Shih et al, 1999; Tao et al, 2003). β2AR can co-immunoprecipitate AKAP150, the rodent homolog of AKAP79 from rat brain lysates (Figure 4A). We hypothesized that the BKCa–β2AR complex might recruit an AKAP due to the constitutive binding of an AKAP to β2AR (Shih et al, 1999; Fraser et al, 2000). In rat brain lysates, AKAP150 could be co-immunoprecipitated with BKCa (Figure 4B), indicating that a BKCa–β2AR–AKAP150 complex exists in native tissue. Figure 4.AKAP150 is associated with β2AR and BKCa channels in brain. (A) AKAP150 immunoblot of immunoprecipitation using β2AR antibody or preimmune serum from rat brain lysates. β2AR specifically associates with AKAP150. (B) BKCa (upper) and AKAP150 (lower) immunoblot of immunoprecipitation using AKAP150 antibody or preimmune serum from rat brain lysates. AKAP150 specifically associates with BKCa. Download figure Download PowerPoint The β2AR–BKCa interaction can be reconstituted in HEK293 cells after expression of both myc-β2AR and BKCa (Figure 5A). β2AR constitutively recruits AKAP79 (Fraser et al, 2000) (Figure 5B), which enables the targeting of AKAP79 to the channel (Figure 5C). Without expression of β2AR, AKAP79 cannot associate with BKCa (Figure 5C), demonstrating the requirement for AKAP79 binding to β2AR in order to assemble the AKAP79–BKCa complex. Overexpression of AKAP79 is not required for β2AR–BKCa interaction (Figure 5A). Likewise, overexpression of BKCa is not required for β2AR–AKAP79 association (Figure 5B). The binding of both AKAP79 and BKCa to β2AR (Figure 5C) is not mutually exclusive, consistent with the findings that BKCa associate with β2AR via the third intracellular loop (i3; Figure 3) and AKAP79 associates with β2AR via the third intracellular loop and the C-terminus independently (Fraser et al, 2000). A mutant AKAP79 (FLAG-AKAP79108–427) that can bind RII and PKA, but cannot associate with β2AR (Fraser et al, 2000), was not recruited into the BKCa complex (Figure 5D). The coupling of β2AR and BKCa was constitutive, as it was not modulated by exposure to isoproterenol (Figure 5E). Collectively, these findings suggest that a functional consequence of β2AR targeting to BKCa may be the facilitation of cAMP-dependent phosphorylation of BKCa by an anchored pool of PKA holoenzyme. Figure 5.β2AR and AKAP79 associate with BKCa channel in HEK293 cells (A) BKCa immunoblot of immunoprecipitation (IP) using anti-myc and preimmune serum from extracts of HEK293 cells overexpressing myc-β2AR and BKCa. β2AR–BKCa association can be reconstituted in HEK293 cells. (B) AKAP79 immunoblot of immunoprecipitation using β2AR antibody and preimmune serum of HEK293 cells overexpressing myc-β2AR and AKAP79. β2AR specifically associates with AKAP79. (C) Coexpression of BKCa, β2AR and AKAP79 (as indicated) in HEK; lysates were immunoprecipitated with preimmune serum, BKCa or β2AR antibodies and blotted with AKAP79 antibody. AKAP79 associates with BKCa, only in cells coexpressing BKCa/β2AR/AKAP79. (D) AKAP79 immunoblot of BKCa immunoprecipitations of extracts from HEK293 cells expressing BKCa/β2AR and either AKAP79 or AKAP79108–427 (not targeted to β2AR). The inability of the truncated AKAP79 to bind to β2AR prevents the assembly of a BKCa–AKAP79 complex. (E) BKCa immunoblot of β2AR immunoprecipitations of β2AR/BKCa-HEK293 cells exposed to isoproterenol. β2AR–BKCa associate in a β-agonist-independent manner. Download figure Download PowerPoint Macromolecular signaling complex enhances β2 agonist activation of BKCa To explore the functional implications of β2AR–BKCa, we coexpressed BKCa, β2AR and AKAP79 in Xenopus oocytes. We recorded channel activity using cell-attached patch clamp and applied a specific β2 agonist, salbutamol (20 μM), either in the recording pipette by back-filling or in the bath solution (20–40 μM) (Chen-Izu et al, 2000). The inclusion of a β2 agonist in the patch pipette increased channel activity over ∼10 min (Figure 6A), consistent with the diffusion of the agonist within the patch pipette and activation of BKCa (P<0.0005 compared to no salbutamol by Wilcoxon's rank sum test). In contrast, channel activity did not increase with bath application of salbutamol, indicating that BKCa is preferentially regulated by β2AR within the channel macromolecular complex, as opposed to those located remotely in the cell (Figure 6B). In a total of 23 patches recorded with salbutamol in the patch pipette, channel activity increased in 21 patches (91%) (Figure 6C), whereas channel activity increased in three of 10 (30%) patches recorded with bath application of salbutamol (P 10%) was observed. Inclusion of salbutamol in the patch pipette significantly increased the number of patches which demonstrated Po increase as compared to bath application. Increased BKCa channel Po was dependent on coexpression of β2AR–AKAP79. (D) Graph of data set summarizing average currents of all 100 sweeps 15 min after salbutamol application. In the box plot, patches without Po increase are excluded. In each box, the mid-line shows the median value, the top and bottom lines show the 75th and 25th percentiles, and the whiskers show the 90th and 10th percentiles. The circles are means of data, including patches without Po increase, and error bars are standard error of means. For experiments on coexpression of BKCa, β2AR and AKAP, the P-value of Wilcoxon's rank sum test <0.0005 between pipette application of salbutamol and no salbutamol (left two circles), and <0.001 between pipette application and bath application of salbutamol (left and right circles). Download figure Download PowerPoint The magnitude of current increase (%) varied extensively (Figure 6D; box plot), an effect that may be due to the variable expression efficiency and/or the time between back-filling the agonist and the formation of the gigaseal. However, the magnitude of current increase was significantly smaller with bath application than that caused by agonist in the patch pipette (Figure 6D; box plot). Although channel activity increased in response to pipette salbutamol in two of five patches (40%) when AKAP79 was not coexpressed, the magnitude of current increase was less than when three components were expressed (Figure 6C and D). These findings suggest that expression of AKAP79 increases the likelihood of salbutamol-mediated modulation (Figure 6C and D). Xenopus oocytes may contain an endogenous AKAP or, alternatively, signaling pathways other than the AKAP-mediated pathway may contribute to the modulation of BKCa. Overall, only expression of all three components of the complex reconstituted full-β2 agonist modulation of the channel (Figure 6D; circles). LTCC associate with BKCa channel through β2AR-dependent scaffold β2ARs in native tissue and expressed in HEK293 cells form SDS-resistant dimers (Figure 7A) (Angers et al, 2000; Salahpour et al, 2003), which associate with BKCa (Figure 7B). Although, G protein-coupled receptors (GPCRs) are thought to function independently as monomers to signal to effector molecules, recent studies have suggested that oligomerization (dimerization) of GPCRs in vivo is a constitutive process, dependent on disulfide bonds and hydrophobic packing (Salahpour et al, 2003), that provides an additional level of functional complexity for their responses (Barki-Harrington et al, 2003). Our findings of β2AR dimerization are consistent with prior reports, although the extent of dimerization seen on SDS–PAGE may not correlate with the extent of dimerization in situ. Figure 7.BKCa channel associates with β2AR dimers. (A) myc immunoblot of β2AR immunoprecipitations from HEK293 cells overexpressing myc-β2AR. The lower molecular form represents the expressed monomeric β2AR, while the higher species represents SDS-resistant β2AR dimers. β2AR antibody specifically immunoprecipitates expressed myc-β2AR. (B) myc immunoblot of BKCa immunoprecipitation from HEK293 cells expressing the indicated constructs. BKCa channels associate with β2AR dimers (the monomer species is obscured by the heavy chain of Ig (HC). Download figure Download PowerPoint As the β2AR can dimerize and associate with BKCa (Figure 7B) and LTCC (Davare et al, 2001), we hypothesized that β2AR might act as a scaffold that not only facilitates cAMP-mediated regulation of the channels but also recruits both ion channels into a macromolecular complex. Despite the observations that Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs) can activate BKCa (Roberts et al, 1990; Gola and Crest, 1993; Nelson et al, 1995; Marrion and Tavalin, 1998; Herrera and Nelson, 2002; Sun et al, 2003), a physical association between the ion channels has not been demonstrated. Immunoprecipitation of LTCC from brain and bladder extract revealed that the channel was part of the BKCa complex (Figure 8A). The association was specific, as the co-immunoprecipitation could be blocked by preabsorption of the antibody with α1c peptide (Figure 8A). Figure 8.β2AR mediates physical colocalization of BKCa and LTCC. (A) BKCa immunoblots of α1c immunoprecipitations from brain and bladder lysates. BKCa (arrow) co-precipitates with α1c (LTCC). Immunoprecipitation specificity was demonstrated using preimmune serum and peptide-blocked antibody (+pep). (B) BKCa immunoblot of α1c immunoprecipitations from HEK293 cells expressing BKCa/β2AR/α1c/β2A. BKCa co-immunoprecipitates with α1c in HEK293 cells coexpressing β2AR. (C) BKCa (left) and α1c/LTCC (right) immunoblot of α1c immunoprecipitates from HEK cells expressing BKCa/β2AR/α1c. BKCa co-precipitates with α1c in a β2a subunit-independent manner. Equivalent expression of α1c in HEK cells is shown (right). (D) BKCa immunoblot of α1c immunoprecipitation of HEK293 cells coexpressing β2AR/BKCa/α1c without β2a subunit. Cells were treated with isoproterenol. α1c-BKCa channels associate in a β-agonist-independent manner. Download figure Download PowerPoint We next examined whether β2AR was required for the assembly of the BKCa–LTCC complex. Coexpression of BKCa, β2AR, and LTCC α1c/β2a subunits reconstituted the association (Figure 8B). The co-immunoprecipitation was blocked by α1c peptide (Figure 8B). The interaction was dependent on β2AR expression because the association was not present in HEK293 cells coexpressing only BKCa and α1c/β2a (Figure 8B). These findings indicate that the assembly of the BKCa–LTCC (α1c+β2a) complex was dependent on β2AR expression. To examine the role of the LTCC β subunit in the assembly of the complex, we coexpressed β2AR, BKCa and α1c in HEK293 cells. The assembly of the BKCa–LTCC complex was dependent on β2AR expression, but not β2a subunit expression (Figure 8C). Exposure of HEK293 cells expressing β2AR, LTCC and BKCa channel to a β agonist (isoproterenol) did not affect the association of BKCa and LTCC (Figure 8D). These findings indicate a novel function for β2AR as a scaffold to permit the physical association of two families of ion channels, which have been previously reported to couple functionally (Figure 9). Figure 9.Molecular model of macromolecular complex depicting the physical and functional regulation of the BKCa channel by β2AR/AKAP79 and α1c. Activation of β2AR by β2 agonist leads to phosphorylation of both BKCa and LTCC, resulting in increased BKCa channel activity. Download figure Download PowerPoint Discussion The present study offers a molecular identification of a mechanism through which BKCa channel activity is specifically regulated by β2AR signaling in brain and smooth muscle. The ability of β2AR to form a complex with BKCa, and concomitantly bind phosphorylation-modulatory components (AKAP79/150) enables specific and local regulation of BKCa channels and defines a signal transduction pathway governing cellular excitability in diverse tissues. The findings also reveal a physiologically important and unanticipated role of β2AR: that of serving as a nonphosphorylation-dependent scaffold to enable regulation of BKCa channels by LTCC. A common theme in signal transduction is the close association of signaling molecules with effectors to enable specific and local regulation (Pawson and Scott, 1997). Specificity of PKA anchoring is achieved by targeting motifs that direct AKAPs to specific cellular sites (Pawson and Scott, 1997). AKAP79/150 has been demonstrated to interact with PKA, PKC and calcineurin (Klauck et al, 1996). The associated PKA and PKC are maintained in an inactive state when bound to AKAP79, but can be activated by cAMP or diacylglycerol (Fau
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