Isoprenaline activates K_ATP current in a PKA-dependent manner.

<p>Representative K_ATP current traces obtained at -60 mV in symmetrical 140 mM K⁺ following the application of isoprenaline (100 nM) in the absence (<b>A</b>) or presence (<b>B</b>) of the active PKA inhibitor peptide (PKAi-RR, 5 μM) in the patch-pipette. Arrows in this and subsequent figures indicate the point at which extracellular [K⁺] was increased from 6 to 140 mM. The current increase in response to isoprenaline is indicated by the dashed lines and arrows. (<b>C</b>) Mean K_ATP current, (normalized to that in 140 mM K⁺) following the application of 100 nM isoprenaline in the absence (n = 6) or presence (n = 8) of PKAi-RR in the patch-pipette (*<i>P</i><0.05; one-way ANOVA, Bonferroni’s <i>post hoc</i> test).</p

Inhibition of PKA attenuates K_v current.

<p>K_v currents were activated in response to 400 ms depolarizing voltage steps from a holding potential of -65 mV. (<b>A</b>) Representative K_v current traces before (control) and after application of 1 μM KT 5720 as indicated. Representative K_v currents shown in this and subsequent figures are in 20 mV increments beginning from -40 mV. (<b>B</b>) Mean (± s.e.m.) I-V plots (normalized to control current at +60 mV) before and after application of 1 μM KT 5720 (n = 8 cells). (<b>C</b>) Representative K_v current traces before and after application of 1 μM KT 5720 obtained in the presence of the PKA inhibitor peptide, PKAi-RR (5 μM) in the patch pipette. (<b>D</b>) Mean (± s.e.m.) I-V plots (normalized to the control currents, <i>i</i>.<i>e</i>. in the presence of PKAi-RR, at +60 mV) before and after application of 1 μM KT 5720 as indicated (n = 5). (<b>E</b>) Representative K_v current traces before and after application of 1 μM KT 5720 in the presence of PKAi-AA (5 μM), an inactive isoform of PKAi-RR, in the patch pipette. (<b>F</b>) Mean (± s.e.m.) I-V plots (normalized to the respective control currents, <i>i</i>.<i>e</i>. in the presence of PKAi-AA at +60 mV) before and after application of 1 μM KT 5720 as indicated (n = 7). *<i>P</i><0.05; two-way ANOVA, Bonferroni’s <i>post hoc</i> test.</p

Regulation of K_v current in MSMCs involves protein phosphatase 2B (PP2B), but not protein phosphatase 2A (PP2A).

<p>(<b>A</b>) Representative K_v current traces obtained from a cell pre-treated with cantharadin (Can, 30 μM) for 10 min before and after application of KT 5720 (1 μM). (<b>B</b>) Mean (± s.e.m.) I-V plots, normalized to current in the presence of cantharadin (30 μM) at +60 mV, before and after the application of KT 5720 (1 μM, n = 4, *<i>P</i><0.05; two-way ANOVA, Bonferroni’s <i>post hoc</i> test). (<b>C</b>) Representative K_v current traces obtained with PP2B/calcineurin auto-inhibitory peptide (CAP, 100 μM) in the patch pipette before and after application of KT 5720 (1 μM). (<b>D</b>) Mean (± s.e.m.) I-V plots, normalized to current in the presence of CAP at +60 mV, before and after application of KT 5720 (1 μM, n = 5). € Mean (± s.e.m.) current at +60 mV (normalized to respective controls) following application of 1 μM KT 5720 in cells pre-treated with cantharadin (Can, 30 μM, n = 4), cyclosporin A (CsA, 4 μM, n = 7), or with CAP (100 μM; n = 5) in the pipette as indicated (*<i>P</i><0.05; one-way ANOVA, Bonferroni’s <i>post hoc</i> test).</p

Inhibition of K_v current by KT 5720 is abolished by disruption of caveolae.

<p>(<b>A</b>) Representative K_v currents following 60 min pre-treatment with 2% methyl-β-cyclodextrin (MβCD) before and after application of 1 μM KT 5720. (<b>B</b>) Mean I-V plots (normalized to control current in the presence of MβCD at +60 mV), before and after application of 1 μM KT 5720 (n = 5). (<b>C</b>) Representative K_v currents in the presence of the caveolin scaffolding-domain peptide (CSDP, 100 μM) in the patch pipette, before and after application of KT 5720 (1 μM). (<b>D</b>) Mean I-V plots (normalized to the current at +60 mV in the presence of CSDP) before and after application of 1 μM KT 5720 (n = 5). (<b>E</b>) Representative K_v currents in the presence of a scrambled version of the caveolin scaffolding-domain peptide (CSDP-scr, 100 μM) in the patch pipette before and after application of KT 5720 (1 μM). (<b>F</b>) Mean I-V plots (normalized to the current at +60 mV in the presence of CSDP-scr) before and after application of 1 μM KT 5720 (n = 6, *<i>P</i><0.05; two-way ANOVA, Bonferroni’s <i>post hoc</i> test).</p

Activation of PKA does not increase K_v current.

<p>(<b>A</b>) Representative K_v current traces obtained before and after application of isoprenaline (1 μM) as indicated. (<b>B</b>) Mean (± s.e.m.) I-V plots (normalized to control current at +40 mV) before and after application of isoprenaline (1 μM; n = 8). (<b>C</b>) Representative K_v current traces as in panel A, but in the presence of IBMX (300 μM) before and after application of isoprenaline (1 μM). (<b>D</b>) Mean I-V plots (normalized to the control current in IBMX at +40 mV) before and after application of isoprenaline in the presence of IBMX (n = 5). (<b>E</b>) Representative K_v current traces before (control) and after application of dibutyryl-cAMP (db-cAMP; 100 μM). (<b>F</b>) Mean (± s.e.m.) I-V plots (normalized to the control current at +40 mV) before and after application db-cAMP (100 μM; n = 4).</p

The adenlylate cyclase inhibitor, dideoxyadenosine attenuates K_v current.

<p>(<b>A</b>) Representative K_v current traces before (control) and after application of 50 μM 2’,5’-dideoxyadenosine (DDA). (<b>B</b>) Mean (± s.e.m.) I-V plots (normalized to control current at +60 mV) before (control) and after application of DDA. *<i>P</i><0.05; two-way ANOVA, Bonferroni’s <i>post hoc</i> test.</p

PKA activation of K_v currents remained following disruption of PKA-AKAP interactions.

<p>(<b>A</b>) Representative K_v current traces obtained immediately after establishing whole-cell recording (< 1 min Ht-31) and 10 min following establishment of the whole-cell configuration with 20 μM Ht-31 in the patch pipette. (<b>B</b>) Mean (± s.e.m.) I-V plots (current density normalized to cell capacitance) immediately after establishing whole-cell recording and 10 min after establishing whole cell configuration in the presence of Ht-31 (20 μM) in the patch pipette (n = 5). (<b>C</b>) Representative K_ATP current traces (normalized to cell capacitance) following the application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 in the patch pipette; the current increase in response to isoprenaline is indicated by the dashed lines and arrow. (<b>D</b>) Mean glibeclamide-sensitive current (normalized to cell capacitance) following application of 100 nM isoprenaline in the absence or presence of 20 μM Ht-31 (n = 8 and 5 cells, respectively; *<i>P</i><0.05; one-way ANOVA, Bonferroni’s <i>post hoc</i> test).</p

Regulatory motifs within the <i>GSTCD/INTS12</i> locus.

<p>The <i>GSTCD</i>/<i>INTS12</i> locus is shown, annotated with RNA sequencing, H3K27Ac histone marks, DNase hypersensitivity, transcription factor binding and CpG islands (UCSC Genome Browser (<a href="http://genome.ucsc.edu/" target="_blank"><u>http://genome.ucsc.edu/</u></a>)) on the Human Feb 2009 (GRCh37/hg19) assembly. For the H3K27Ac histone marks and RNA sequence tracks, peak height is proportional to signal amplitude, with colours representing datasets in different cell backgrounds (pale blue H3K27Ac histone trace = human umbilical vein endothelial cell (HUVEC); blue/grey = K562 erythroleukaemia cells). For the DNase hypersensitivity and transcription factor binding tracks, a grey band indicates the extent of the hypersensitive region and the intensity of the band is proportional to the maximum signal strength observed in any cell line.</p

Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see Table S5 for patient demographics).

<p>Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone.0074630.s009" target="_blank">Table S5</a> for patient demographics).</p

<i>GSTCD</i> and <i>INTS12</i> gene expression is altered following exposure of HASM cells to TGFβ1.

<p>Human airway smooth muscle (HASM) cells were exposed to 10ng/ml TGFβ1 for 4 or 24 hours. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=5). Significant increases in both <i>GSTCD</i> and <i>INTS12</i> gene expression were observed following 24h exposure to TGFβ1 (<i>P</i><0.05 <i>GSTCD</i>, <i>P</i><0.01 <i>INTS12</i>) and after 4h TGFβ1 exposure in <i>INTS12</i> expression (<i>P</i><0.05).</p