Understanding the interactions that occur between KCNE1 and KCNQ1 : stoichiometry, gating and dynamic movements

The IKs current has an important role in repolarizing the cardiac action potential. KCNQ1 subunits form a tetrameric voltage-gated potassium channel, with which an accessory beta-subunit, KCNE1, can interact. KCNE1 binding to KCNQ1 remarkably alters channel kinetics by delaying activation, increasing current density and removing inactivation. There has always been confusion about how many KCNE1 subunits can associate with the KCNQ1 tetramer. Several groups have reported a strict fixed stoichiometry of two KCNE1 subunits to four KCNQ1 subunits. However, others have shown that the ratio varies depending on the concentration of KCNE1 subunits available. Using whole cell and single channel patch clamp recordings of tethered fusion constructs with different ratios of KCNE1:KCNQ1, as well as photo-crosslinking experiments, we show that up to four KCNE1 subunits can associate with the complex. Therefore, in vitro, IKs can have a variable stoichiometry. In further photo-crosslinking experiments we show that two adjacent residues in KCNE1 interact with KCNQ1 in different channel states, open and closed. Additionally, we show that these interactions are likely taking place with the pore domain of the channel. We also confirm what other groups had proposed, that KCNE1 moves within the cleft of KCNQ1 during channel activation, since the rates of crosslinking change when the channels are held at more depolarized holding potentials. Finally, we investigate whether or not all four voltage sensors have to activate and the complex undergo a concerted step in order for the pore to conduct current. We employ a mutation, E160R, which applies electrostatic repulsion to the positive charges of the voltage sensor and keeps it in a resting conformation. By locking one, two, three and four voltage sensors down we show, via whole cell and single channel recordings, that the channel can conduct when only one voltage sensor is free to move. From these experiments, we provide additional evidence that the IKs channel gates in an allosteric fashion, where each additional voltage sensor movement results in a progressively higher open probability. Additionally, we propose that there is cooperativity between KCNQ1 subunits, where movement of one voltage sensor facilitates movement of a neighboring voltage sensor.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

cAMP-dependent regulation of IKs single-channel kinetics

International audienceThe delayed potassium rectifier current, IKs, is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3′-5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance–voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation

β-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2

Acute phosphatidylinositol 4,5 bisphosphate depletion destabilizes sarcolemmal expression of cardiac L-type Ca2+ channel CaV1.2

CaV1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through Gq-coupled AT1 receptors, angiotensin II triggers a decrease in PIP2, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP2 depletion suppresses CaV1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that CaV1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP2 stabilizes CaV1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP2 depletion and destabilization of CaV1.2 expression. We tested this hypothesis and report that CaV1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular CaV1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP2 supplementation. Functional data revealed acute angiotensin II reduced CaV1.2 currents and Ca2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP2 stabilizes CaV1.2 membrane lifetimes, and angiotensin II-induced PIP2 depletion destabilizes sarcolemmal CaV1.2, triggering their removal, and the acute reduction of CaV1.2 currents and contractility

BIN1 knockdown rescues systolic dysfunction in aging male mouse hearts

Cardiac dysfunction is a hallmark of aging in humans and mice. Here we report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the aging phenotype. Our data indicate that age-associated overexpression of BIN1 occurs alongside dysregulated endosomal recycling and disrupted trafficking of cardiac CaV1.2 and type 2 ryanodine receptors. These deficiencies affect channel function at rest and their upregulation during acute stress. In vivo echocardiography reveals reduced systolic function in old mice. BIN1 knockdown using an adeno-associated virus serotype 9 packaged shRNA-mBIN1 restores the nanoscale distribution and clustering plasticity of ryanodine receptors and recovers Ca2+ transient amplitudes and cardiac systolic function toward youthful levels. Enhanced systolic function correlates with increased phosphorylation of the myofilament protein cardiac myosin binding protein-C. These results reveal BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium