Inhibition of the heterotetrameric K⁺ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

Figures & data

Figure 1. Co-expression of AMPK decreased voltage-gated outward current in KCNQ1/KCNE1 expressing Xenopus oocytes. (A) Original tracings of the current induced by depolarization from −60 mV to −40, −20, 0, 20, 40, 60, and 80 mV in Xenopus oocytes injected with water (a), expressing KCNQ1/KCNE1 without (b) or with (c) additional co-expression of wild type AMPK, of kinase dead mutant ^αK45RAMPK (d) or of constitutively active ^γR70QAMPK (e). (B) Arithmetic means ± SEM (n = 22–36) of depolarization-induced K⁺ current at +80 mV in Xenopus oocytes injected with water (1^st bar), expressing KCNQ1/KCNE1 without (2^nd bar) or with additional coexpression of wild type AMPK (3^rd bar), of kinase dead mutant ^K45RAMPK (4^th bar) or of constitutively active ^R70QAMPK (5^th bar). ***(p < 0.001) indicates statistically significant difference from the values obtained in oocytes expressing KCNQ1/KCNE1 alone. (C) Arithmetic means ± SEM of depolarization-induced current (I_g) as a function of the potential in Xenopus oocytes injected as in B. (D) Arithmetic means ± SEM (n = 12–24) of depolarization-induced K⁺ current at +80 mV in Xenopus oocytes injected with water (1^st bar), expressing KCNQ1/KCNE1 without (2^nd bar) or with additional co-expression of constitutively active ^R70QAMPK (3^rd bar). The oocytes were incubated in the absence (white bars) or presence of 10 μM AMPK inhibitor compound C (black bars) for the indicated number of hours prior to the experiment. ***(p < 0.001) indicates statistically significant difference from the values obtained in oocytes expressing KCNQ1/KCNE1 alone. ^{#, ###} (p < 0.05, p < 0.001) indicate significant difference from the absence of compound C. (E) Arithmetic means ± SEM (n = 8–17) of depolarization-induced K⁺ current at +80 mV in Xenopus oocytes injected with water (1^st bar), expressing KCNQ1/KCNE1 without (2^nd bar) or with additional coexpression of constitutively active ^R70QAMPKα2 (3^rd bar). ^### (p < 0.001) indicates significant difference from the absence of ^R70QAMPKα2.

Figure 2. Co-expression of AMPK did not significantly modify activation kinetics of voltage-gated outward currents in KCNQ1/KCNE1-expressing Xenopus oocytes. Arithmetic means ± SEM (n = 31–36) of the time constant (τ) plotted vs. the depolarizing potential in Xenopus oocytes expressing KCNQ1/KCNE1 alone (closed symbols) or KCNQ1/KCNE1 together with constitutively active ^R70QAMPK (open symbols).

Figure 3. Co-expression of AMPK decreased the KCNQ1 protein abundance within the plasma membrane of oocytes. (A) Confocal images of KCNQ1 protein abundance in the plasma membrane of Xenopus oocytes injected with water (2^nd upper panel), expressing KCNQ1/KCNE1 without (3^rd upper panel) or with additional co-expression of wild type AMPK (1^st lower panel), of kinase dead mutant ^αK45RAMPK (2^nd lower panel) or of constitutively-active ^γR70QAMPK (3^rd lower panel). The cells were subjected to immunofluorescent staining using FITC-conjugated antibody (grey/green). The 1^st upper panel serves as control (absence of primary antibody). (B) Original Western Blots of total KCNQ1 (upper panel) and GAPDH (lower panel) in Xenopus oocytes injected with water (1^st lane), expressing KCNQ1/KCNE1 without (2^nd lane) or with additional co-expression of wild-type AMPK (3^rd lane), of kinase dead mutant ^αK45RAMPK (4^th lane) or of constitutively-active ^γR70QAMPK (5^th lane). The lower bar diagram displays the densitometric analysis of the Western blots (arithmetic means ± SEM [n = 3]).

Figure 4. Similar to ^γR70QAMPK the ubiquitin ligase Nedd4-2 downregulated KCNQ1/KCNE1. (A) Original tracings of the current induced by depolarization from −60 mV to −40, −20, 0, 20, 40, 60, and 80 mV in Xenopus oocytes injected with water (a), expressing KCNQ1/KCNE1 without (b,d) or with (c,e) additional coexpression of constitutively active ^γR70QAMPK in the absence (b,c) or presence (d,e) of Nedd4-2. (B) Arithmetic means ± SEM (n = 10–19) of depolarization-induced current at +80 mV in Xenopus oocytes injected with water (H₂O), expressing KCNQ1/KCNE1 (Q1E1) without or with additional co-expression of constitutively active ^R70QAMPK (AMPK^R70Q) in the absence (open bars) or presence (closed bars) of Nedd4-2. *** (p < 0.001) indicates statistically significant difference from the values obtained in oocytes expressing KCNQ1/KCNE1 alone. (C) Arithmetic means ± SEM (n = 11–14) of depolarization-induced current at +80 mV in Xenopus oocytes injected with KCNQ1/KCNE1 without (white bars) or with additional coexpression of constitutively active ^R70QAMPK (black bars) in the absence (2 left bars) or presence of Nedd4-2 (2 middle bars) or of Nedd4-2^S795A (2 right bars). *** (p < 0.001) indicates statistically significant difference from the values obtained in oocytes expressing KCNQ1/KCNE1 alone. # (p < 0.05) indicates statistically significant difference from the absence of ^R70QAMPK.

Figure 5. Similar to ^γR70QAMPK the ubiquitin ligase Nedd4-2 decreased the KCNQ1 protein abundance in the cell membrane. Confocal images of KCNQ1 protein abundance in the plasma membrane of Xenopus oocytes expressing KCNQ1/KCNE1 without (1^st panel) or with additional coexpression of constitutively active ^γR70QAMPK (2^nd panel), of the ubiquitin ligase Nedd4-2 (3^rd panel) or of both, ^γR70QAMPK and Nedd4-2 (4^th panel). The cells were subjected to immunofluorescent staining using FITC-conjugated antibody (grey/green).

Figure 6. Depolarization of proximal renal tubule cells by stimulation of AMPK (A) Original Western Blots demonstrating expression of AMPKα (upper panel) and KCNQ1 (lower panel; GAPDH was used as loading control) in isolated proximal tubules of C57 BL/6 mice. (B) Arithmetic means ± SEM (n = 7–8) of the potential difference across the basolateral membrane (PD) of isolated perfused proximal renal tubules from C57 BL/6 mice incubated for 1 h at 22°C in the absence (left bar) or presence (right bar) of AMPK stimulator AICAR (10 mM). * (p < 0.05) indicates statistically significant difference from the values obtained in absence of AICAR.