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. 2023 Aug 31;12:e83466. doi: 10.7554/eLife.83466

Figure 2. Differential functional effects of nano-Cα targeted to either Q1 or E1 on IKs.

(A) Cartoon showing Q1/E1-YFP complex co-expressed with or without free protein kinase A (PKA) Cα subunit. (B) Representative immunoblots of lysates from HEK293 cells co-expressing Q1/E1-YFP with either empty pcDNA3.1 vector or free Cα. Anti-pKCNQ1 (top) detects phosphorylated KCNQ1-S27, anti-KCNQ1 (middle) detects total KCNQ1, and anti-actin (bottom) detects total actin. N=1. (C) IKs activation curves in Chinese hamster ovary (CHO) cells co-expressing Q1, E1-YFP with either empty pcDNA3.1 vector (black symbols, n=13) or free PKA Cα (red symbols, n=13). (D) Tail-decay times for currents recorded from cells co-expressing Q1/E-YFP+yotiao and either nano or free PKA Cα, or cells co-expressing Q1[S27A]/E1-YFP+yotiao and free PKA Cα (p=0.0532, one-way ANOVA). (E–H) Cartoon, immunoblots, IKs activation curves, and population current densities of Q1/E1-YFP complex expressed with either nano (n=10) or nanoCα (n=10). (I) Cartoon showing targeting of nanoCα to Q1/E1 complex via YFP tag on Q1 C-terminus. (J) Exemplar IKs traces from CHO cells co-expressing Q1-YFP/E1 with either nanoCα (left) or catalytically inactive nanoCα [T198A] mutant (right). (K) Population current densities (nano, n=26; nanoCα, n=19; nanoCα[T198A], n=10).

Figure 2—source data 1. Differential functional effects of nano-Cα targeted to either Q1 or E1 on IKs.
Figure 2—source data 2. Differential functional effects of nano-Cα targeted to either Q1 or E1 on IKs.

Figure 2.

Figure 2—figure supplement 1. Evidence that nanoCα but not free protein kinase A (PKA) Cα is recruited to E1-YFP in the Q1/E1-YFP channel complex.

Figure 2—figure supplement 1.

Representative immunoblots of lysates from untransfected HEK293 cells (UT) or co-expressing Q1/E1-YFP with either nanoCα or free Cα, immunoprecipitated with anti-PKA Cα and probed with (A) anti-YFP to detect E1-YFP, or (B) anti-PKA Cα to detect nanoCα and free Cα, respectively. (C) Input controls from same samples blotted with anti-YFP. (D) Anti-actin loading controls. N=2 for each blot.
Figure 2—figure supplement 1—source data 1. Full immunoblots of experiments showing nanoCα but not free PKA Cα is recruited to E1-YFP in the Q1/E1-YFP channel complex.
Figure 2—figure supplement 2. NanoCα targeted to the C-terminus of TASK1 via a GFP tag does not inhibit K+ current.

Figure 2—figure supplement 2.

(A) Exemplar current-voltage relationship elicited by a ramp stimulus (–120 to +60 mV) in a Chinese hamster ovary (CHO) cell expressing hTASK1-GFP+nano. (B) Exemplar current-voltage relationship elicited by a ramp stimulus in a CHO cell expressing hTASK1-GFP+nanoCα. (C) Population current density at 0 mV for cells expressing hTASK1-GFP with either nano (black bar, n=7) or nanoCα (red bar, n=9).
Figure 2—figure supplement 2—source data 1. NanoCα targeted to the C-terminus of TASK1 via a GFP tag does not inhibit K+ current.