Cecile Terrenoire

p11, an annexin II subunit, an auxiliary protein associated with the background K+ channel, TASK-1

TASK‐1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage‐ and Ca2+‐sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK‐1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C‐terminal amino acids, Ser‐Ser‐Val, in TASK‐1. Using series of C‐terminal TASK‐1 deletion mutants and several TASK‐1–GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK‐1 to the plasma membrane. p11 association with the TASK‐1 channel masks an endoplasmic reticulum retention signal identified as Lys‐Arg‐Arg that precedes the Ser‐Ser‐Val sequence.

Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics

Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na+ channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na+ channel current (INaL) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of INaL such that increasing the pacing rate markedly reduces INaL and, in addition, increases its inhibition by the Na+ channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband’s iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.

The Cardiac IKs Potassium Channel Macromolecular Complex Includes the Phosphodiesterase PDE4D3

The cardiac IKs potassium channel is a macromolecular complex consisting of α-(KCNQ1) and β-subunits (KCNE1) and the A kinase-anchoring protein (AKAP) Yotiao (AKAP-9), which recruits protein kinase A) and protein phosphatase 1 to the channel. Here, we have tested the hypothesis that specific cAMP phosphodiesterase (PDE) isoforms of the PDE4D family that are expressed in the heart are also part of the IKs signaling complex and contribute to its regulation by cAMP. PDE4D isoforms co-immunoprecipitated with IKs channels in hearts of mice expressing the IKs channel. In myocytes isolated from these mice, IKs was increased by pharmacological PDE inhibition. PDE4D3, but not PDE4D5, co-immunoprecipitated with the IKs channel only in Chinese hamster ovary cells co-expressing AKAP-9, and PDE4D3, but not PDE4D5, co-immunoprecipitated with AKAP-9. Functional experiments in Chinese hamster ovary cells expressing AKAP-9 and either PDE4D3 or PDE4D5 isoforms revealed modulation of the IKs response to cAMP by PDE4D3 but not PDE4D5. We conclude that PDE4D3, like protein kinase A and protein phosphatase 1, is recruited to the IKs channel via AKAP-9 and contributes to its critical regulation by cAMP.

The bee venom peptide tertiapin underlines the role of IKACh in acetylcholine-induced atrioventricular blocks

Acetylcholine (ACh) is an important neuromodulator of cardiac function that is released upon stimulation of the vagus nerve. Despite numerous reports on activation of IKACh by acetylcholine in cardiomyocytes, it has yet to be demonstrated what role this channel plays in cardiac conduction. We studied the effect of tertiapin, a bee venom peptide blocking IKACh, to evaluate the role of IKACh in Langendorff preparations challenged with ACh. ACh (0.5 μM) reproducibly and reversibly induced complete atrioventricular (AV) blocks in retroperfused guinea‐pig isolated hearts (n=12). Tertiapin (10 to 300 nM) dose‐dependently and reversibly prevented the AV conduction decrements and the complete blocks in unpaced hearts (n=8, P<0.01). Tertiapin dose‐dependently blunted the ACh‐induced negative chronotropic response from an ACh‐induced decrease in heart rate of 39±16% in control conditions to 3±3% after 300 nM tertiapin (P=0.01). These effects were not accompanied by any significant change in QT intervals. Tertiapin blocked IKACh with an IC50 of 30±4 nM with no significant effect on the major currents classically associated with cardiac repolarisation process (IKr, IKs, Ito1, Isus, IK1 or IKATP) or AV conduction (INa and ICa(L)). In summary, tertiapin prevents dose‐dependently ACh‐induced AV blocks in mammalian hearts by inhibiting IKACh.

Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an IKs transgenic mouse

Inherited gain‐of‐function mutations of genes coding for subunits of the heart slow potassium (IKs) channel can cause familial atrial fibrillation (AF). Here we consider a potentially more prevalent mechanism and hypothesize that β‐adrenergic receptor (β‐AR)‐mediated regulation of the IKs channel, a natural gain‐of‐function pathway, can also lead to AF. Using a transgenic IKs channel mouse model, we studied the role of the channel and its regulation by β‐AR stimulation on atrial arrhythmias. In vivo administration of isoprenaline (isoproterenol) predisposes IKs channel transgenic mice but not wild‐type (WT) littermates that lack IKs to prolonged atrial arrhythmias. Patch‐clamp analysis demonstrated expression and isoprenaline‐mediated regulation of IKs in atrial myocytes from transgenic but not WT littermates. Furthermore, computational modelling revealed that β‐AR stimulation‐dependent accumulation of open IKs channels accounts for the pro‐arrhythmic substrate. Our results provide evidence that β‐AR‐regulated IKs channels can play a role in AF and imply that specific IKs deregulation, perhaps through disruption of the IKs macromolecular complex necessary for β‐AR‐mediated IKs channel regulation, may be a novel therapeutic strategy for treating this most common arrhythmia.

Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry

Non‐technical summary  The human heart is a pump that works only when its internal electrical system coordinates both its filling and its capacity to eject blood. This critical electrical timing is coordinated by many different ion channels, and this study looks at the one known as IKs. Mutations in its α subunit, KCNQ1, constitute the majority of cases of the disorder long QT syndrome (LQT‐1). Here we have examined properties of human cardiac cells during very early stages of development and found evidence for the manner in which the subunits of IKs assemble; our data suggest that this assembly may be flexible and may change during development and/or disease.