Mechanism of PKCε regulation of cardiac GIRK channel gating

Figures

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  Fig. 1.. PKCε activation alters the action potential in the optical mapping of mouse hearts. A, Fluorescence images of the mapped field. SVC; superior vena cava, RAA; right atrial appendage, RV; right ventricle, Stim; bipolar stimulation electrode. Heat maps cover the RAA area only in the middle panels and represent the APD (APD60) maps after 25 min of perfusion with vehicle (DMSO) (Top) or 10 μM DCP-LA (Bottom). The Right panels are the APD60 maps after administration of 200 nM tertiapin-Q. B, the bar graphs show that in DMSO-treated hearts, tertiapin-Q did not significantly prolong the APD60 (N.S., paired Student’s t test, N = 3 hearts). In DCP-LA-treated hearts, tertiapin-Q significantly prolonged the APD60 (*P < 0.05, paired Student’s t test, N = 4 hearts). C is a quantification of the percent of APD60 change after tertiapin-Q administration, which significantly increased the APD60 in DCP-LA-pretreated hearts (*P < 0.05, one sample t test, N = 4), but not in DMSO pretreatment (N.S. one sample t test, N = 3).

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  Fig. 2.. PKCε activation alters action potential parameters in human iPSC atrial cardiomyocytes. Human-induced pluripotent stem cell differentiated into atrial cardiomyocytes were treated with 100 µM DCP-LA for 90 min prior to patch-clamp electrophysiology experiments. A, Spontaneous action potentials were elicited under current-clamp conditions and APD50 was measured in control and DCP-LA treated cardiomyocytes (****P < 0.00005 using Student’s unpaired t test; n = 6 to 7 cells per condition). B, Tertiapin-Q sensitive potassium currents were assessed in control and DCP-LA-treated cells under voltage-clamp conditions (**P < 0.005 using Student’s unpaired t test). C, Representative IKACh currents from atrial cardiomyocytes. Data are current densities of n = 5 cells per condition). C, Voltage ramp protocol and representative traces of GIRK channel currents in control and DCP-LA-treated conditions are shown.

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  Fig. 3.. PKCε enhances the activity of GIRK4-containing channels. A, TEVC experiments with GIRK4*S143T (GIRK4*) RNA co-expressed in Xenopus oocytes along with the muscarinic M2 receptor with (red bars/triangles) or without the catalytic subunit of PKCε (black bars/triangles). B, Representative traces. C, Whole-cell patch-clamp experiments with GIRK4WT channels expressed in HEK293T cells with or without the catalytic subunit of PKCε. D, Representative traces of patch-clamp experiments. E, TEVC experiments with GIRK1/4 RNA co-expressed in Xenopus oocytes along with the muscarinic M2 receptor with (red bars/triangles) or without the catalytic subunit of PKCε (black bars/triangles). F, Representative traces with or without PKCε. G, Whole-cell patch-clamp experiments with GIRK1/4 channels expressed in HEK293T cells with or without the catalytic subunit of PKCε. H, Representative traces of patch-clamp experiments. Basal GIRK activity in mammalian cells was evaluated in response to 140 mM K+ solution. Patch-clamp data are whole-cell current densities expressed as mean ± SD (n = 5 cells per condition). Basal GIRK activity in oocytes was evaluated in response to 96 mM K+ solution and…

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  Fig. 4.. A C-terminal serine residue of GIRK4 is responsible for the stimulatory effects of PKCε. A, TEVC experiments with GIRK4WT and GIRK4(S148A) RNA co-expressed in Xenopus oocytes along with the muscarinic M2 receptor with (red bars/circles) or without the catalytic subunit of PKCε (black bars/triangles). B, Representative traces of WT and mutant GIRK4 channels with and without PKCε. C, TEVC experiments with GIRK4WT and GIRK4(S418E) RNA co-expressed in Xenopus oocytes along with the muscarinic M2 receptor with (red bars/triangles) or without the catalytic subunit of PKCε (black bars/circles). D, Representative traces of WT and mutant GIRK4 channels with and without PKCε. Basal GIRK activity in oocytes was evaluated in response to 96 mM K+ solution and agonist-induced activity was evaluated in response to ACh. Data are whole-oocyte currents expressed as mean ± SD (n = 8-15 cells per condition). Negative currents indicate inward flow of positively charged ions; **P < 0.05, **P < 0.005, ****P < 0.00005 calculated using one-way ANOVA with Tukey’s post hoc test for A and Dunnett’s post hoc test for C.

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  Fig. 5.. PKCε enhances GIRK1/4 channel activity by strengthening channel–PIP2 interactions. A, This shows the scheme for optogenetic depletion of PIP2 using mCherry-CRY2-5-ptaseOCRL. HEK293T cells were transfected with GIRK1, GIRK4, GFP-CIBN-CAAX, mCherry-CRY2-5-ptaseOCRL. B and C, Representative traces showing GIRK1/4 current depletion with (C) and without (B) the presence of PKCε. D, GIRK1/4 current remaining after PIP2 depletion is greater when PKCε is present. E, The kinetics of current depletion (tau) following PIP2 depletion are slower when PKCε is present indicating greater affinity of the phosphorylated channel for PIP2. F, The 5-ptaseOCRL-mediated decrease in current is characterized by mono-exponential fits in the presence and absence of PKCε. Data are currents recorded from HEK293T cells using patch-clamp in whole-cell mode and are shown as mean ± SD (n = 8 to10 cells per group). Statistical significance was calculated using Students’ t test, *P < 0.05, **P < 0.005.

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  Fig. 6.. S418 is a critical residue in mediating the effects of PKCε on GIRK4. HEK293T cells were transfected with GIRK4 or GIRK4(S418A), GFP-CIBN-CAAX, mCherry-CRY2-5-ptaseOCRL. A–C, Representative traces showing GIRK1/4 current depletion with (B) and without (A) the presence of PKCε and C, GIRK4(S418A) with PKCε. D, GIRKWT current remaining after PIP2 depletion is greater when PKCε is present, unlike GIRK4(S418A). E, 5-ptaseOCRL-mediated decrease in current is characterized by mono-exponential fits in the presence and absence of PKCε. F, The kinetics of current depletion (tau) following PIP2 depletion are slower when PKCε is present indicating greater affinity of the phosphorylated channel for PIP2. Data are currents recorded from HEK293T cells using patch-clamp in whole-cell mode and are shown as mean ± SD (n = 5 to 8 cells per group). Statistical significance was calculated using one-way ANOVA and Tukey’s post hoc test, *P < 0.05, **P < 0.005.