Alain Coulombe

Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure.

Abstract. Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 m KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system.

SAP97 and Dystrophin Macromolecular Complexes Determine Two Pools of Cardiac Sodium Channels Nav1.5 in Cardiomyocytes

Rationale: The cardiac sodium channel Nav1.5 plays a key role in excitability and conduction. The 3 last residues of Nav1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin–dystrophin complex. As dystrophin is absent at the intercalated discs, Nav1.5 could potentially interact with other, yet unknown, proteins at this site. Objective: The aim of this study was to determine whether Nav1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results: Immunostaining experiments demonstrated that Nav1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Nav1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Nav1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Nav1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (INa) measured by patch-clamp. The INa generated by Nav1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Nav1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions: These data support a model with at least 2 coexisting pools of Nav1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.

MOG1: A New Susceptibility Gene for Brugada Syndrome

Background— Brugada syndrome (BrS) is caused mainly by mutations in the SCN5A gene, which encodes the &agr;-subunit of the cardiac sodium channel Nav1.5. However, ≈20% of probands have SCN5A mutations, suggesting the implication of other genes. MOG1 recently was described as a new partner of Nav1.5, playing a potential role in the regulation of its expression and trafficking. We investigated whether mutations in MOG1 could cause BrS. Methods and Results— MOG1 was screened by direct sequencing in patients with BrS and idiopathic ventricular fibrillation. A missense mutation p.Glu83Asp (E83D) was detected in a symptomatic female patient with a type-1 BrS ECG but not in 281 controls. Wild type (WT)- and mutant E83D-MOG1 were expressed in HEK Nav1.5 stable cells and studied using patch-clamp assays. Overexpression of WT-MOG1 alone doubled sodium current (INa) density compared to control conditions (P<0.01). In contrast, overexpression of mutant E83D alone or E83D+WT failed to increase INa (P<0.05), demonstrating the dominant-negative effect of the mutant. Microscopy revealed that Nav1.5 channels failed to properly traffic to the cell membrane in the presence of the mutant. Silencing endogenous MOG1 demonstrated a 54% decrease in INa density. Conclusions— Our results support the hypothesis that dominant-negative mutations in MOG1 can impair the trafficking of Nav1.5 to the membrane, leading to INa reduction and clinical manifestation of BrS. Moreover, silencing MOG1 reduced INa, demonstrating that MOG1 is likely to be important in the surface expression of Nav1.5 channels. All together, our data support MOG1 as a new susceptibility gene for BrS.

Angiotensin II Signaling Pathways Mediate Expression of Cardiac T-Type Calcium Channels

Abstract— Recent studies indicate that cardiac T-type Ca2+ current (ICaT) reappears in hypertrophied ventricular cells. The aim of this study was to investigate the role of angiotensin II (Ang II), a major inducer of cardiac hypertrophy, in the reexpression of T-type channel in left ventricular hypertrophied myocytes. We induced cardiac hypertrophy in rats by abdominal aorta stenosis for 12 weeks and thereafter animals were treated for 2 weeks with losartan (12 mg/kg per day), an antagonist of type 1 Ang II receptors (AT1). In hypertrophied myocytes, we showed that the reexpressed ICaT is generated by the CaV3.1 and CaV3.2 subunits. After losartan treatment, ICaT density decreased from 0.40±0.05 pA/pF (n=26) to 0.20±0.03 pA/pF (n=27, P <0.01), affecting CaV3.1- and CaV3.2-related currents. The amount of CaV3.1 mRNA increased during hypertrophy and retrieved its nonhypertrophic level after losartan treatment, whereas the amount of CaV3.2 mRNA was unaffected by stenosis. In cultured newborn ventricular cells, chronic Ang II application (0.1 &mgr;mol/L) also increased ICaT density and CaV3.1 mRNA amount. UO126, a mitogen-activated protein kinase kinase-1/2 (MEK1/2) inhibitor, reduced Ang II–increased ICaT density and CaV3.1 mRNA amount. Bosentan, an endothelin (ET) receptor antagonist, reduced Ang II–increased ICaT density without affecting the amount of CaV3.1 mRNA. Finally, cotreatment with bosentan and UO126 abolished the Ang II–increased ICaT density. Our results show that AT1-activated MEK pathway and autocrine ET-activated independent MEK pathway upregulate T-type channel expression. Ang II–increased of ICaT density observed in hypertrophied myocytes may play a role in the pathogenesis of Ca2+ overload and arrhythmias seen in cardiac pathology.

RNA Interference Targeting STIM1 Suppresses Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat

Our objective was to study the expression and function of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum protein recently identified as the calcium sensor that regulated Ca(2+)-released activated channels in T cells. STIM1 was found to be upregulated in serum-induced proliferating human coronary artery smooth muscle cells (hCASMCs) as well as in the neointima of injured rat carotid arteries. Growth factors-induced proliferation was significantly lower in hCASMC transfected with STIM1 siRNA than in those transfected with scrambled siRNA (increase relative to 0.1% S: 116 +/- 12% and 184 +/- 16%, respectively, P < 0.01). To assess the role of STIM1 in preventing vascular smooth muscle cells (VSMCs) proliferation in vivo, we infected balloon-injured rat carotid arteries with an adenoviral vector expressing a short hairpin (sh) RNA against rat STIM1 mRNA (Ad-shSTIM1). Intima/media ratios reflecting the degree of restenosis were significantly lower in Ad-shSTIM1- infected arteries than in Ad-shLuciferase-infected arteries (0.34 +/- 0.02 vs. 0.92 +/- 0.11, P < 0.006). Finally, we demonstrated that silencing STIM1 prevents activation of the transcription factor NFAT (nuclear factor of activated T cell). In conclusion, STIM1 appears as a major regulator of in vitro and in vivo VSMC proliferation, representing a novel and original pharmacological target for prominent vascular proliferative diseases.

Kv4 Potassium Channels Form a Tripartite Complex With the Anchoring Protein SAP97 and CaMKII in Cardiac Myocytes

Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, Ito, in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited Ito in cardiac myocytes, whereas its overexpression by using an adenovirus increased Ito. Kv4.3 channels without the SAL sequence were no longer regulated by Ca2+/calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels.

Dynamic of Ion Channel Expression at the Plasma Membrane of Cardiomyocytes

Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.

Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes

SummaryCa2+- and Ba2+-permeable channel activity from adult rat ventricular myocytes, spontaneously appeared in the three single-channel recording configurations: cell-attached, and excised inside-out or outside-out membrane patches. Single-channel activity was recorded at steady-state applied membrane potentials including the entire range of physiologic values, and displayed no “rundown” in excised patches. This activity occurred in irregular bursts separated by quiescent periods of 5 to 20 min in cell-attached membrane patches, whereas in excised patch experiments, this period was reduced to 2 to 10 min. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. Three conductance levels: 22, 45 and 78 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. These channels were significantly permeable to divalent cations and showed little or no permeability to potassium or sodium ions. The inorganic blockers of voltage-gated Ca channels, cobalt (2mm), cadmium (0.5mm) or nickel (3mm), had no apparent effect on these spontaneous unitary currents carried by barium ions. Under 10−5m bay K 8644 or nitrendipine, the activity was clearly increased in about half of the tested excised inside-out membrane patches. Both dihydropyridines enhanced openings of the larger conductance level, which was only very occasionally seen under control conditions. When the single-channel activity became sustained under 5×10−6m Bay K 8644, it was possible to calculate the mean unitary current at different membrane potentials and show that the mean current value increased with membrane potential.

Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes

Cholesterol is an important determinant of cardiac electrical properties. However, underlying mechanisms are still poorly understood. Here, we examine the hypothesis that cholesterol modulates the turnover of voltage-gated potassium channels based on previous observations showing that depletion of membrane cholesterol increases the atrial repolarizing current IKur. Whole-cell currents and single-channel activity were recorded in rat adult atrial myocytes (AAM) or after transduction with hKv1.5-EGFP. Channel mobility and expression were studied using fluorescence recovery after photobleaching (FRAP) and 3-dimensional microscopy. In both native and transduced-AAMs, the cholesterol-depleting agent MβCD induced a delayed (≈7 min) increase in IKur; the cholesterol donor LDL had an opposite effect. Single-channel recordings revealed an increased number of active Kv1.5 channels upon MβCD application. Whole-cell recordings indicated that this increase was not dependent on new synthesis but on trafficking of existing pools of intracellular channels whose exocytosis could be blocked by both N-ethylmaleimide and nonhydrolyzable GTP analogues. Rab11 was found to coimmunoprecipitate with hKv1.5-EGFP channels and transfection with Rab11 dominant negative (DN) but not Rab4 DN prevented the MβCD-induced IKur increase. Three-dimensional microscopy showed a decrease in colocalization of Kv1.5 and Rab11 in MβCD-treated AAM. These results suggest that cholesterol regulates Kv1.5 channel expression by modulating its trafficking through the Rab11-associated recycling endosome. Therefore, this compartment provides a submembrane pool of channels readily available for recruitment into the sarcolemma of myocytes. This process could be a major mechanism for the tuning of cardiac electrical properties and might contribute to the understanding of cardiac effects of lipid-lowering drugs.

Ionic basis of the action potential prolongation in ventricular myocytes from Syrian hamsters with dilated cardiomyopathy

OBJECTIVE The aim of our study was to determine the main electrophysiological alterations associated with cardiac dilation in MS200 strain Syrian hamsters, a model of genetically determined cardiomyopathy. METHODS Ventricular action potentials (APs) were recorded with standard microelectrodes in isolated hearts from 120-day-old cardiomyopathic (strain MS200) and age-matched control (strain CHF148) Syrian hamsters. Ionic currents were recorded from single ventricular myocytes using the whole-cell patch-clamp technique. RESULTS In MS200, AP was prolonged and the plateau phase was markedly increased as compared to CHF148. Differences in both AP duration and 4-aminopyridine-induced AP lengthening between epicardial and endocardial tissues were less marked in MS200 than in CHF148 ventricles. Cell size and membrane capacitance were not higher in MS200 than in CHF148 myocytes, indicating the absence of cell hypertrophy in myopathic ventricles. The L-type calcium current (ICa,L) density was significantly reduced in MS200 and the voltage-dependence of both steady-state activation and inactivation was altered. The voltage-dependent outward current was composed of both transient (Ito1) and sustained (Iss) components, respectively sensitive and insensitive to 4-aminopyridine. Ito1 density was strongly depressed in MS200 compared to CHF148, whereas Iss density was only slightly reduced. The conductance-voltage and steady-state inactivation relationships for Ito1 were shifted to more positive potentials in MS200. The Ito1 recovery process was markedly slower in MS200 than in CHF148. The steady-state current-voltage relationships, in the physiological voltage range, were superimposable in MS200 and CHF148. CONCLUSIONS In ventricular myocytes from dilated heart of MS200 Syrian hamsters, Ito1 is more drastically depressed than ICa,L. Such an observation might partially explain dilation-induced AP lengthening.