Filamin A heart valve disease as a genetic cause of inherited bicuspid and tricuspid aortic valve disease.

OBJECTIVE: Variants in the FLNA gene have been associated with mitral valve dystrophy (MVD), and even polyvalvular disease has been reported. This study aimed to analyse the aortic valve and root involvement in FLNA-MVD families and its impact on outcomes. METHODS: 262 subjects (37 (18-53) years, 140 male, 79 carriers: FLNA+) from 4 FLNA-MVD families were included. Echocardiography was performed in 185 patients and histological analysis in 3 explanted aortic valves. The outcomes were defined as aortic valve surgery or all-cause mortality. RESULTS: Aortic valve alterations were found in 58% of FLNA+ compared with 6% of FLNA- (p<0.001). 9 (13.4%) FLNA+ had bicuspid aortic valve compared with 4 (3.4%) FLNA- (p=0.03). Overall, the transvalvular mean gradient was slightly increased in FLNA+ (4.8 (4.1-6.1) vs 4.0 (2.9-4.9) mm Hg, p=0.02). The sinuses of Valsalva and sinotubular junction diameters were enlarged in FLNA+ subjects (all p<0.05). 8 FLNA+ patients underwent aortic valve surgery (0 in relatives; p<0.001). Myxomatous remodelling with an infiltration of immune cells was observed. Overall survival was similar between FLNA+ versus FLNA- subjects (86±5% vs 85±6%, p=0.36). There was no statistical evidence for an interaction between genetic status and sex (p=0.15), but the survival tended to be impaired in FLNA+ men (p=0.06) whereas not in women (p=0.71). CONCLUSION: The patients with FLNA variants present frequent aortic valve disease and worse outcomes. Bicuspid aortic valve is more frequent in patients carrying the FLNA-MVD variants. These unique features should be factored into the management of patients with dystrophic and/or bicuspid aortic valve.

Genetics and pathophysiology of mitral valve prolapse.

Mitral valve prolapse (MVP) is a common condition affecting 2-3% of the general population, and the most complex form of valve pathology, with a complication rate up to 10-15% per year in advanced stages. Complications include mitral regurgitation which can lead to heart failure and atrial fibrillation, but also life-threatening ventricular arrhythmia and cardiovascular death. Sudden death has been recently brought to the forefront of MVP disease, increasing the complexity of management and suggesting that MVP condition is not properly understood. MVP can occur as part of syndromic conditions such as Marfan syndrome, but the most common form is non-syndromic, isolated or familial. Although a specific X-linked form of MVP was initially identified, autosomal dominant inheritance appears to be the primary mode of transmission. MVP can be stratified into myxomatous degeneration (Barlow), fibroelastic deficiency, and Filamin A-related MVP. While FED is still considered a degenerative disease associated with aging, myxomatous MVP and FlnA-MVP are recognized as familial pathologies. Deciphering genetic defects associated to MVP is still a work in progress; although FLNA, DCHS1, and DZIP1 have been identified as causative genes in myxomatous forms of MVP thanks to familial approaches, they explain only a small proportion of MVP. In addition, genome-wide association studies have revealed the important role of common variants in the development of MVP, in agreement with the high prevalence of this condition in the population. Furthermore, a potential genetic link between MVP and ventricular arrhythmia or a specific type of cardiomyopathy is considered. Animal models that allow to advance in the genetic and pathophysiological knowledge of MVP, and in particular those that can be easily manipulated to express a genetic defect identified in humans are detailed. Corroborated by genetic data and animal models, the main pathophysiological pathways of MVP are briefly addressed. Finally, genetic counseling is considered in the context of MVP.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

AIMS: Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. METHODS AND RESULTS: Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-ß and inflammation in the disease. CONCLUSION: The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Obinutuzumab plus fludarabine and cyclophosphamide in previously untreated, fit patients with chronic lymphocytic leukemia: a subgroup analysis of the GREEN study.

GREEN (NCT01905943) is a nonrandomized, open-label, single-arm, phase 3b study investigating the safety and efficacy of obinutuzumab alone or in combination with chemotherapy in chronic lymphocytic leukemia (CLL). We report the preplanned subgroup analysis of 140 previously untreated, fit CLL patients who received obinutuzumab plus fludarabine and cyclophosphamide (G-FC). The primary endpoint was safety and tolerability. Efficacy was the secondary endpoint. Obinutuzumab 1000 mg was administered intravenously on Day (D)1 (dose split D1‒2), D8 and D15 of Cycle (C)1, and D1 of C2-6 (28-day cycles). Standard intravenous/oral doses of fludarabine and cyclophosphamide were administered on D1-3 of C1-6. Overall, 87.1% of patients experienced grade ≥ 3 adverse events (AEs), including neutropenia (67.1%) and thrombocytopenia (17.1%). Serious AEs were experienced by 42.1% of patients. Rates of grade ≥ 3 infusion-related reactions and infections were 19.3% and 15.7%, respectively. Overall response rate was observed in 90.0%, with 46.4% of patients achieving complete response (CR; including CR with incomplete marrow recovery). Minimal residual disease negativity rates were 64.3% in peripheral blood and 35.7% in bone marrow (intent-to-treat analysis). After a median observation time of 25.6 months, 2 year progression-free survival was 91%. Frontline G-FC represents a promising treatment option for fit patients with CLL.

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia.

Mitral valve diseases affect ∼3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from nonsyndromic mitral valve dysplasia (MVD). The filamin A (FLNA) protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD-causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function has mostly remained elusive. In this study, using NMR spectroscopy and interaction assays, we investigated FLNA-MVD-causing V711D and H743P mutations. Our results clearly indicated that both mutations almost completely destroyed the folding of the FLNA5 domain, where the mutation is located, and also affect the folding of the neighboring FLNA4 domain. The structure of the neighboring FLNA6 domain was not affected by the mutations. These mutations also completely abolish FLNA's interactions with protein tyrosine phosphatase nonreceptor type 12, which has been suggested to contribute to the pathogenesis of FLNA-MVD. Taken together, our results provide an essential structural and molecular framework for understanding the molecular bases of FLNA-MVD, which is crucial for the development of new therapies to replace surgery.

Non-syndromic Mitral Valve Dysplasia Mutation Changes the Force Resilience and Interaction of Human Filamin A.

Filamin A (FLNa), expressed in endocardial endothelia during fetal valve morphogenesis, is key in cardiac development. Missense mutations in FLNa cause non-syndromic mitral valve dysplasia (FLNA-MVD). Here, we aimed to reveal the currently unknown underlying molecular mechanism behind FLNA-MVD caused by the FLNa P637Q mutation. The solved crystal structure of the FLNa3-5 P637Q revealed that this mutation causes only minor structural changes close to mutation site. These changes were observed to significantly affect FLNa's ability to transmit cellular force and to interact with its binding partner. The performed steered molecular dynamics simulations showed that significantly lower forces are needed to split domains 4 and 5 in FLNA-MVD than with wild-type FLNa. The P637Q mutation was also observed to interfere with FLNa's interactions with the protein tyrosine phosphatase PTPN12. Our results provide a crucial step toward understanding the molecular bases behind FLNA-MVD, which is critical for the development of drug-based therapeutics.

Safety of obinutuzumab alone or combined with chemotherapy for previously untreated or relapsed/refractory chronic lymphocytic leukemia in the phase IIIb GREEN study.

The safety of obinutuzumab, alone or with chemotherapy, was studied in a non-randomized, open-label, non-comparative, phase IIIb study (GREEN) in previously untreated or relapsed/refractory chronic lymphocytic leukemia. Patients received obinutuzumab 1000 mg alone or with chemotherapy (investigator's choice of fludarabine-cyclophosphamide for fit patients, chlorambucil for unfit patients, or bendamustine for any patient) on days 1, 8 and 15 of cycle 1, and day 1 of cycles 2-6 (28-day cycles), with the cycle 1/day 1 dose administered over two days. The primary end point was safety/tolerability. Between October 2013 and March 2016, 972 patients were enrolled and 971 treated (126 with obinutuzumab monotherapy, 193 with obinutuzumab-fludarabine-cyclophosphamide, 114 with obinutuzumab-chlorambucil, and 538 with obinutuzumab-bendamustine). Grade ≥3 adverse events occurred in 80.3% of patients, and included neutropenia (49.9%), thrombocytopenia (16.4%), anemia (9.6%), and pneumonia (9.0%); rates were similar in first-line and relapsed/refractory patients, and in first-line fit and unfit patients. Using expanded definitions, infusion-related reactions were observed in 65.4% of patients (grade ≥3, 19.9%; mainly seen during the first obinutuzumab infusion), tumor lysis syndrome in 6.4% [clinical and laboratory; highest incidence with obinutuzumab-bendamustine (9.3%)], and infections in 53.7% (grade ≥3, 20.1%). Serious and fatal adverse events were seen in 53.1% and 7.3% of patients, respectively. In first-line patients, overall response rates at three months post treatment exceeded 80% for all obinutuzumab-chemotherapy combinations. In the largest trial of obinutuzumab to date, toxicities were generally manageable in this broad patient population. Safety data were consistent with previous reports, and response rates were high. (clinicaltrials.gov identifier: 01905943).

Genetics of syndromic and non-syndromic mitral valve prolapse.

Mitral valve prolapse (MVP) is a common condition that affects 2%-3% of the general population. MVP is thought to include syndromic forms such as Marfan syndrome and non-syndromic MVP, which is the most frequent form. Myxomatous degeneration and fibroelastic deficiency (FED) are regarded as two different forms of non-syndromic MVP. While FED is still considered a degenerative disease associated with ageing, frequent familial clustering has been demonstrated for myxomatous MVP. Familial and genetic studies led to the recognition of reduced penetrance and large phenotypic variability, and to the identification of prodromal or atypical forms as a part of the complex spectrum of the disease. Whereas autosomal dominant mode is the common inheritance pattern, an X linked form of non-syndromic MVP was recognised initially, related to Filamin-A gene, encoding for a cytoskeleton protein involved in mechanotransduction. This identification allowed a comprehensive description of a new subtype of MVP with a unique association of leaflet prolapse and paradoxical restricted motion in diastole. In autosomal dominant forms, three loci have been mapped to chromosomes 16p11-p12, 11p15.4 and 13q31-32. Although deciphering the underlying genetic defects is still a work in progress, DCHS1 mutations have been identified (11p15.4) in typical myxomatous disease, highlighting new molecular pathways and pathophysiological mechanisms leading to the development of MVP. Finally, a large international genome-wide association study demonstrated the implication of frequent variants in MVP development and opened new directions for future research. Hence, this review focuses on phenotypic, genetic and pathophysiological aspects of MVP.

New insights into mitral valve dystrophy: a Filamin-A genotype-phenotype and outcome study.

Aims: Filamin-A (FLNA) was identified as the first gene of non-syndromic mitral valve dystrophy (FLNA-MVD). We aimed to assess the phenotype of FLNA-MVD and its impact on prognosis. Methods and results: We investigated the disease in 246 subjects (72 mutated) from four FLNA-MVD families harbouring three different FLNA mutations. Phenotype was characterized by a comprehensive echocardiography focusing on mitral valve apparatus in comparison with control relatives. In this X-linked disease valves lesions were severe in men and moderate in women. Most men had classical features of mitral valve prolapse (MVP), but without chordal rupture. By contrast to regular MVP, mitral leaflet motion was clearly restricted in diastole and papillary muscles position was closer to mitral annulus. Valvular abnormalities were similar in the four families, in adults and young patients from early childhood suggestive of a developmental disease. In addition, mitral valve lesions worsened over time as encountered in degenerative conditions. Polyvalvular involvement was frequent in males and non-diagnostic forms frequent in females. Overall survival was moderately impaired in men (P = 0.011). Cardiac surgery rate (mainly valvular) was increased (33.3 ± 9.8 vs. 5.0 ± 4.9%, P < 0.0001; hazard ratio 10.5 [95% confidence interval: 2.9-37.9]) owing mainly to a lifetime increased risk in men (76.8 ± 14.1 vs. 9.1 ± 8.7%, P < 0.0001). Conclusion: FLNA-MVD is a developmental and degenerative disease with complex phenotypic expression which can influence patient management. FLNA-MVD has unique features with both MVP and paradoxical restricted motion in diastole, sub-valvular mitral apparatus impairment and polyvalvular lesions in males. FLNA-MVD conveys a substantial lifetime risk of valve surgery in men.

Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension.

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

MVP-Associated Filamin A Mutations Affect FlnA-PTPN12 (PTP-PEST) Interactions.

Although the genetic basis of mitral valve prolapse (MVP) has now been clearly established, the molecular and cellular mechanisms involved in the pathological processes associated to a specific mutation often remain to be determined. The FLNA gene (encoding Filamin A; FlnA) was the first gene associated to non-syndromic X-linked myxomatous valvular dystrophy, but the impacts of the mutations on its function remain un-elucidated. Here, using the first repeats (1-8) of FlnA as a bait in a yeast two-hybrid screen, we identified the tyrosine phosphatase PTPN12 (PTP-PEST) as a specific binding partner of this region of FlnA protein. In addition, using yeast two-hybrid trap assay pull down and co-immunoprecipitation experiments, we showed that the MVP-associated FlnA mutations (G288R, P637Q, H743P) abolished FlnA/PTPN12 interactions. PTPN12 is a key regulator of signaling pathways involved in cell-extracellular matrix (ECM) crosstalk, cellular responses to mechanical stress that involve integrins, focal adhesion transduction pathways, and actin cytoskeleton dynamics. Interestingly, we showed that the FlnA mutations impair the activation status of two PTPN12 substrates, the focal adhesion associated kinase Src, and the RhoA specific activating protein p190RhoGAP. Together, these data point to PTPN12/FlnA interaction and its weakening by FlnA mutations as a mechanism potentially involved in the physiopathology of FlnA-associated MVP.

Developmental basis for filamin-A-associated myxomatous mitral valve disease.

AIMS: We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. METHODS AND RESULTS: Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. CONCLUSIONS: These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.

Filamin-a-related myxomatous mitral valve dystrophy: genetic, echocardiographic and functional aspects.

Myxomatous dystrophy of the cardiac valves is a heterogeneous group of disorders, including syndromic diseases such as Marfan syndrome and isolated valvular diseases. Mitral valve prolapse, the most common form of this disease, is presumed to affect approximately 2% to 3% of the population and remains one of the most common causes of valvular surgery. During the past years, important effort has been made to better understand the pathophysiological basis of mitral valve prolapse. Autosomal-dominant transmission is the usual inheritance with reduced penetrance and variable expressivity. Three loci have been mapped to chromosomes 16p11-p12, 11p15.4 and 13q31-32, but the underlying genetic defects are not currently known. An X-linked recessive form has been originally described by Monteleone and Fagan in 1969. Starting from one large French family and three smaller other families in which MVP was transmitted with an X-linked pattern, we have been able to identify three filamin A mutations p.Gly288Arg and p.Val711Asp and a 1,944-bp genomic deletion coding for exons 16 to 19. In this review, we describe the genetic, echocardiographic and functional aspects of the filamin-A-related myxomatous mitral valve dystrophy.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) stabilizes the open pore conformation of the Kv11.1 (hERG) channel.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a phospholipid that has been shown to modulate several ion channels, including some voltage-gated channels like Kv11.1 (hERG). From a biophysical perspective, the mechanisms underlying this regulation are not well characterized. From a physiological perspective, it is critical to establish whether the PIP(2) effect is within the physiological concentration range. Using the giant-patch configuration of the patch-clamp technique on COS-7 cells expressing hERG, we confirmed the activating effect of PIP(2). PIP(2) increased the hERG maximal current and concomitantly slowed deactivation. Regarding the molecular mechanism, these increased amplitude and slowed deactivation suggest that PIP(2) stabilizes the channel open state, as it does in KCNE1-KCNQ1. We used kinetic models of hERG to simulate the effects of the phosphoinositide. Simulations strengthened the hypothesis that PIP(2) is more likely stabilizing the channel open state than affecting the voltage sensors. From the physiological aspect, we established that the sensitivity of hERG to PIP(2) comes close to that of KCNE1-KCNQ1 channels, which lies in the range of physiological PIP(2) variations.

Delayed rectifier K(+) currents and cardiac repolarization.

The two components of the cardiac delayed rectifier current have been the subject of numerous studies since firstly described. This current controls the action potential duration and is highly regulated. After identification of the channel subunits underlying IKs, KCNQ1 associated with KCNE1, and IKr, HERG, their involvement in human cardiac channelopathies have provided various models allowing the description of the molecular mechanisms of the KCNQ1 and HERG channels trafficking, activity and regulation. More recently, studies have been focusing on the unveiling of different partners of the pore-forming proteins that contribute to their maturation, trafficking, activity and/or degradation, on one side, and on their respective expression in the heterogeneous cardiac tissue, on the other side. The aim of this review is to report and discuss the major works on IKs and IKr and the most recent ones that help to understand the precise function of these currents in the heart.

The 19th Ion Channel Meeting. September 2008, France.

The annual meeting of the French Ion Channels Society, held on the Mediterranean coast of France, is aimed at gathering the international scientific community working on various aspects of ion channels. In this report of the 19th edition of the meeting, held in September 2008, we summarize selected symposia on aspects of the ion channel field from fundamental to clinical research. The meeting is an opportunity for leading investigators as well as young researchers to present and discuss their recent advances and future challenges in the ion channel field.

IKs response to protein kinase A-dependent KCNQ1 phosphorylation requires direct interaction with microtubules.

AIMS: KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K(+) channel responsible for I(Ks)-a major repolarizing current in the human heart-and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view. METHODS AND RESULTS: Using a yeast two-hybrid screening, we detected a direct interaction between beta-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of beta-tubulin and KCNQ1 in transfected COS-7 cells and in guinea pig cardiomyocytes. Using immunocytochemistry, we also found that they co-localized in cardiomyocytes. We tested the effects of microtubule-disrupting and -stabilizing agents (colchicine and taxol, respectively) on the KCNQ1-KCNE1 channel activity in COS-7 cells by means of the permeabilized-patch configuration of the patch-clamp technique. None of these agents altered I(Ks). In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the I(Ks) response to protein kinase A (PKA)-mediated stimulation depended on microtubule polymerization in COS-7 cells and in cardiomyocytes. Strikingly, KCNQ1 channel and Yotiao phosphorylation by PKA-detected by phospho-specific antibodies-was maintained, as was the association of the two partners. CONCLUSION: We propose that the KCNQ1-KCNE1 channel directly interacts with microtubules and that this interaction plays a major role in coupling PKA-dependent phosphorylation of KCNQ1 with I(Ks) activation.

NHE-RF1 protein rescues DeltaF508-CFTR function.

In cystic fibrosis (CF), the DeltaF508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of DeltaF508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study for CF treatment. Recent studies have demonstrated that PDZ-containing proteins play an essential role in determining polarized plasma membrane expression of ionic transporters. In the present study we have hypothesized that the PDZ-containing protein NHE-RF1, which binds to the carboxy terminus of CFTR, rescues DeltaF508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding DeltaF508-CFTR and NHE-RF1 were intranuclearly injected in A549 or Madin-Darby canine kidney (MDCK) cells, and DeltaF508-CFTR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with DeltaF508-CFTR alone presented a low chloride channel activity, whereas its coexpression with NHE-RF1 significantly increased both the basal and forskolin-activated chloride conductances. This last effect was lost with DeltaF508-CFTR deleted of its 13 last amino acids or by injection of a specific NHE-RF1 antisense oligonucleotide, but not by NHE-RF1 sense oligonucleotide. Immunocytochemical analysis performed in MDCK cells transiently transfected with DeltaF508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of DeltaF508-CFTR but not that of a trafficking defective mutant potassium channel (KCNQ1). These data demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-RF1, can rescue DeltaF508-CFTR activity.