Abstract-The slow I KS K ϩ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca 2ϩ -CaM that prevents inactivation, facilitates channel assembly, and mediates a Ca 2ϩ -sensitive increase of I KS-current, with a considerable Ca 2ϩ -dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or I KS channels with a Ca 2ϩ -insensitive CaM mutant markedly suppresses the currents and produces a right shift in the voltage-dependence of channel activation. KCNE1 association to KCNQ1 long-QT mutants significantly improves mutant channel expression and prevents macroscopic inactivation. However, the marked right shift in channel activation and the subsequent decrease in current amplitude cannot restore normal levels of I KS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca 2ϩ -sensitive I KS -current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias. Key Words: KCNQ Ⅲ potassium channels Ⅲ Kv7 Ⅲ calmodulin Ⅲ KCNE Ⅲ long QT K CNQ channels represent a family of voltage-gated K ϩ channels (Kv7) that plays a major role in brain and cardiac excitability. 1,2 Mutations of human KCNQ genes lead to severe cardiovascular and neurological disorders such as the cardiac long-QT syndrome (LQT) and neonatal epilepsy. Coassembly of KCNQ1 with KCNE1 ␤ subunits produces the I KS -current that is crucial for repolarization of the cardiac action potential. [3][4][5] The cytoplasmic KCNQ C-termini were shown to feature 4 ␣ helices. 6 We previously identified the last ␣ helix of the C terminus (helix D, aa.589 -620) as a region important for the tetrameric assembly of KCNQ1 ␣ subunits. 7 This region also binds Yotiao, an A-kinase-anchoring protein that targets PKA on the I KS channel complex. 8 The first 2␣ helices of KCNQ1-5 form a calmodulin-binding domain (CBD), including an IQ motif that mediates Ca 2ϩ -free calmodulin (apoCaM) binding. 6,9 Although KCNQ channels bind calmodulin (CaM), the role of CaM in channel function remains controversial. Recent studies found a role for CaM as a Ca 2ϩ -sensor of KCNQ2/4/5 channels, 10,11 whereas others suggested a role in channel assembly. 9 So far, no information has been available about the interaction of calmodulin with cardiac I KS channels and its pathophysiological impact to KCNQ1-related LQT channnelopathies. Here, we show that LQT mutations located near the IQ motif of KCNQ1 C terminus impair ...

SummaryThe SWI/SNF complex plays an important role in mouse embryonic stem cells (mESCs), but it remains to be determined whether this complex is required for the pluripotency of human ESCs (hESCs). Using RNAi, we demonstrated that depletion of BRG1, the catalytic subunit of the SWI/SNF complex, led to impaired self-renewing ability and dysregulated lineage specification of hESCs. A unique composition of the BRG1-SWI/SNF complex in hESCs was further defined by the presence of BRG1, BAF250A, BAF170, BAF155, BAF53A, and BAF47. Genome-wide expression analyses revealed that BRG1 participated in a broad range of biological processes in hESCs through pathways different from those in mESCs. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) demonstrated that BRG1 played a repressive role in transcriptional regulation by modulating the acetylation levels of H3K27 at the enhancers of lineage-specific genes. Our data thus provide valuable insights into molecular mechanisms by which transcriptional repression affects the self-renewal and differentiation of hESCs.

Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regularities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker, no correlation between V(1/2) and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental findings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE β-subunits.

Our findings indicate that glycine52 in the transmembrane domain is critical for KCNE1 function. The mutant G52R-KCNE1 has a dominant negative effect on I(ks) current, which reduces the I(ks) current amplitude and leads to a prolongation of the cardiac action potential. This could underlie the molecular mechanism of ventricular arrhythmias and sudden death in those patients.