The auxiliary subunit KCNE1 regulates KCNQ1 channel response to sustained calcium-dependent PKC activation

Fig 1. KCNE1 subunit is critical for KCNQ1 channel internalization. (A) Top, Representative confocal images of live HEK cells expressing KCNQ1-GFP with or without KCNE1 subunit after sustained cPKC activation (1 μM, cPKC activator pseudo RACK1, 90 min), and cPKC inhibition (1 μM, Go6976, 90 min). The same amount of vector pcDNA3.1(+) plasmid was used in place of KCNE1 in experiments of KCNQ1 alone. Bottom, the fluorescence profiles of the yellow boxed areas in images in top panels. M: cytoplasmic membrane; C: cytoplasm. The dotted green line marks the background fluorescence. (B) Summary data of normalized membrane localization of KCNQ1 in experiments conducted above. Scale bars, 5 μm. Control = vehicle. * p<0.05, (n = the number of cells).

Fig 2. KCNQ1/KCNE1 channels internalized after sustained cPKC stimulation regardless of their stoichiometry. (A), Representative confocal images of HEK cells expressing KCNQ1-GFP and KCNE1 at different ratios as indicated, under sustained cPKC stimulation (1 μM cPKC activator peptide KAC1-1, 90 min). (B), Summary data of normalized KCNQ1-GFP membrane localization. Control = control peptide C1. Scale bars, 5 μm. *p<0.05, (n = number of cells).

Fig 3. Serine 102 of KCNE1 is involved in IKs internalization after cPKC stimulation. (A) Left, Representative confocal images of HEK cell expressing KCNQ1-GFP and KCNE1 or KCNE1(S102A) and after sustained cPKC stimulation (cPKC activator peptide KAC1-1, 1 μM, 90 min). Right, Summary data of normalized KCNQ1 membrane localization. (B) Left, Representative IKs current traces of HEK cells expressing KCNQ1 and KCNE1 or KCNQ1 and KCNE1(S102A) after cPKC stimulation (cPKC activator peptide KAC1-1, 1 μM, 90 min). Right, Summary data of normalized current density at +40 mV in experiments conducted to the left. (C) Left, Representative confocal images of HEK cells expressing KCNQ1-GFP and KCNE1 or KCNE1(S102A) together with DN-PKCβII or CA-PKCβII. Right, Summary data of KCNQ1 membrane localization. (D) Left, Representative IKs current traces of HEK cells expressing KCNQ1 and KCNE1 or KCNQ1 and KCNE1(S102A) together with DN-PKCβII or CA-PKCβII. Right, Summary data of current density at +40 mV in experiments conducted to the left. Control = control peptide C1 in Panel A and B. Scale bars, 5 μm. *p<0.05, (n = number of cells).

Fig 4. KCNE1(S102A) mutation prevents cPKC-dependent IKs channel internalization in isolated adult rat ventricular myocytes. Top, Representative images of cardiomyocytes expressing adenovirus of KCNQ1-GFP and KCNE1 or KCNE1(S102A), with sustained α1-AR stimulation (Phe, 30 μM, 90 min), and enlarged yellow boxed areas. Bottom, Summary data of normalized membrane localization of KCNQ1 from experiments conducted as in top panel. Control = vehicle. Scale bars, 5 μm. *p<0.05, n = number of cells measured.