BKCa channel is phosphorylated by associated PKA. (A) Autoradiograph (left) of BKCa channel immunoprecipitated from brain incubated with the catalytic subunit of PKA (PKA_c) and [γ-³²P]ATP and size-fractionated on SDS–8% PAGE; the specificity was established using preimmune serum and PKA inhibitor, PKI_5–24. 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 PKA_c or cAMP±PKI. cAMP activates an associated, endogenous kinase that is inhibited by PKI, indicating that PKA_c 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 PKA_c from the BKCa macromolecular complex, as indicated by the reduction of cAMP-induced phosphorylation in the pretreated lane (+).

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.

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.

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.

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 AKAP79^108–427 (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.

Localized upregulation of BKCa channels via β2AR signaling. (A, B) Representative current traces of BKCa channels coexpressed with β2AR and AKAP79 in a Xenopus oocyte. Channel activity was recorded using cell-attached patch clamp during 20 ms, +150 mV test pulses from −50 mV, and then returned to −50 mV. Salbutamol (20 μM) was applied in the patch pipette by back-filling (A) or in the bath (B). A total of 100 sweeps were applied at various times as indicated. Sweeps 10, 40 and 80 at each time are shown. ‘c' represents closed state of the channel. Average currents of all 100 sweeps are shown at the bottom at the indicated times (min) after the GΩ seal formation (A) or bath application of salbutamol (B). (C) Total number of patches that have been recorded versus the number of patches in which an increased open probability of BKCa (P_o increase >10%) was observed. Inclusion of salbutamol in the patch pipette significantly increased the number of patches which demonstrated P_o increase as compared to bath application. Increased BKCa channel P_o 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 P_o 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 P_o 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).

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).

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/β_2A. 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 β_2a 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.

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.