Treiber F , Rosker C , Keren-Raifman T , Steinecker B , Gorischek A , Dascal N , Schreibmayer W .

P 22974 Austrian Science Fund FWF

	Fig. 1. Homooligomeric GIRK4 channels are regulated by PKA.A.: Original current recording derived from an oocyte injected with cRNA encoding GIRK4 and m2R. Membrane potential was kept constant at − 80 mV. Basal current (IHK) was induced by changing from the physiological extracellular medium (ND96) to a medium containing 96 mmole/L K+ (HK). G-protein activation was achieved by perfusion with HK containing 10− 5 mole/L acetylcholine. An injection needle was inserted during agonist activation. In order to activate PKA 3 pmole cAMP were injected into the cytosol of the oocyte (both insertion and injection are indicated by an arrow). After washout of agonist and HK, both IHK and IACh were elicited again in order to demonstrate the increase of currents through GIRK4 complexes.B.: Statistical analysis of the effect of injection of SpCAMPS, RpCAMPS and water on agonist induced currents through homooligomeric GIRK4S143T complexes. Number of individual oocytes tested in parenthesis above each bar. **: the mean value deviates statistically significant from both RpCAMPS and H2O at the p < 0.01 level.C.: Statistical analysis of the effect of cAMP injection on basal currents and agonist induced currents through homooligomeric and heterooligomeric GIRK channel complexes. Number of individual oocytes tested in parenthesis above each bar. *, (***): the mean value deviates statistically significant from zero at the p < 0.05 (0.001) level.
	Fig. 2. Phosphorylation of the cytosolic regions of GIRK4 by PKA in-vitro.A.: upper panel: Autoradiogram showing 32P incorporation of the GIRK4 C- and N-terminal fusion proteins following incubation with PKA-cs and ATP-γ-32P. Lower panel: Coomassie stain of the electrophoresis gel shown in upper panel.B: Statistical analysis of 32P incorporation into N- and C-terminus. The number of individual phosphorylation experiments is shown in parenthesis above each bar. ***: the mean value deviates statistically significant from GST at the p < 0.001 level.
	Fig. 3. Role of T199 and S412 in the heterologous facilitation of homooligomeric GIRK4⁎ channels.A.: Original current recording derived from an oocyte injected with cRNA encoding GIRK4⁎T199CS412C and m2R. Membrane potential was kept constant at − 80 mV. After induction of basal current (IHK) by superfusion with HK and G-protein activation by 10− 5 mole/L acetylcholine 5 pmole cAMP were injected into the cytosol oocyte (injection is indicated by an arrow).B.: Statistical analysis of the effect of cAMP injection on agonist induced currents through GIRK4⁎WT and mutant channels. Number of individual oocytes tested in parenthesis above each bar. ***: the mean value deviates statistically significant from GIRK4⁎WT at the p < 0.001 level.
	Fig. 4. Effect of T199 and S412 on PKA-cs catalysed 32P incorporation into the GIRK4 C-terminus.A.: Upper panel: Autoradiogram showing the effect of T199 and S412 on 32P incorporation into the GIRK4 C-terminal fusion protein (upper panel). Lower panel: Coomassie stain of the electrophoresis gel shown in upper panel.B.: Statistical analysis of 32P incorporation into WT and mutant forms of the GIRK4 C-terminus. The number of individual phosphorylation experiments is shown in parenthesis above each bar. ***: the mean value deviates statistically significant from zero at the p < 0.001 level. §§§: the mean value deviates statistically significant from WT at the p < 0.001 level.
	Fig. 5. Role of individual subunits in the heterologous facilitation of heterooligomeric IK,ACh channels.A: Original current recording derived from an oocyte injected with cRNA encoding GIRK1WT, GIRK4WT and m2R. Membrane potential was kept constant at − 80 mV. After induction of basal current (IHK) by superfusion with HK, 4 pmole cAMP were injected into the cytosol of the oocyte (both insertion of needle and injection are indicated by an arrow).B: similar to 5 A, but the oocyte was injected with cRNA encoding the phosphorylation deficient GIRK1S385CS401CT407C and GIRK4T199CS412C subunits.C: Statistical analysis of the effect of cAMP injection on basal and agonist induced currents through heterooligomeric GIRK1/GIRK4 WT and mutant channel complexes. Number of individual oocytes tested in parenthesis above each bar. *, (**): the mean value deviates significantly from zero at the p < 0.05 (0.01) level. §, (§§§): the mean value deviates statistically significant from GIRK1WT/GIRK4WT at the p < 0.05 (0.001) level.
	Fig. 6. Primary structure alignment of the four GIRK subunit isoforms at the protein level.S/Ts in the GIRK4 sequence are highlighted in grey (italics: no major effect on phosphorylation in-vitro; italics/bold: major effect on phosphorylation in-vitro, but no functional consequence) or black (both profound effect on phosphorylation in-vitro and on heterologous facilitation). S/Ts that have been found to be important for heterologous facilitation in the GIRK1 sequence are also highlighted in black [25]. S191 that has been reported to be responsible for PKC regulation is marked in darker grey (both GIRK1 and GIRK4; [36]). Arrows indicate the truncations of the C-terminus used for phosphorylation in vitro (suppl. Figs. S1). TM1, TM2 and P denote the trans membrane and the pore helices, respectively.

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