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

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

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

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

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

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

Cellular localization of M₁-cameleons transiently expressed in HEK293 cells.

<p>HEK293 cells were transiently transfected with M₁-cam1 (<b>A</b>), M₁-cam2 (<b>B</b>), M₁-cam3 (<b>C</b>), M₁-cam4 (<b>D</b>) or M₁-cam5 (<b>E</b>). Images were acquired by confocal microscopy and show fluorescence emission at >530 nm following excitation at 514 nm. Scale bar, 15 µm.</p

Overview of M₁ AChR chimeric constructs.

<p>Primers used for introducing <i>AgeI</i> site into the i3 loop are shown where applicable.</p

MCh-induced changes in FRET in HEK293 M₁-cam5 cells.

<p>HEK293 cells stably expressing M₁-cam5 were observed using fluorescence imaging with single wavelength excitation (436 nm) and dual wavelength emission (436 nm to detect ECFPc and 535 nm to detect EYFP^46L. <b>A</b>. Representative images showing plasma membrane distribution of ECFPc (480 nm, left panel) and EYFP^46L (535 nm, middle panel), which overlap (right panel) as expected for signals from the same population of receptors. <b>B–D</b>, right panels: blue and yellow traces represent signals from ECFP and EYFP, respectively; left panels: red traces represent the FRET signal (ratio of F_EYFP/F_ECFP). Addition of MCh (100 µM) induced decreases in FRET, which remained constant throughout the application period (30–40 s; <b>B</b>); this effect was reversed on addition of atropine (1 µM; <b>C</b>); and the MCh-induced change in FRET ratio could be completely prevented by pre-addition of atropine (<b>D</b>). FRET data have been normalized so that the initial FRET signal is 100%. Emission traces are expressed as the change in fluorescence intensity from the basal fluorescence level (<i>F</i>/<i>F</i>₀). Representative traces of at least three independent experiments are shown.</p

Comparisons of maximal FRET changes and rate constants for a variety of orthosteric and allosteric ligands in HEK293-M₁-cam5 cells.

<p>Cells were stimulated with a maximally effective concentration of each agonist and FRET changes (<b>A</b>) and K<i>_obs</i> values (<b>B</b>) were determined as described above. Data are presented as means ± s.e.m. from at least three independent experiments. One-way AVOVA (*<i>p</i><0.05; **<i>p</i><0.005; ***<i>p</i><0.0001).</p