Psychometric properties of Clinical Learning Environment Inventory and its association with Moroccan nursing students' satisfaction: A PLS-SEM approach.

Background: The Clinical Learning Environment (CLE) is integral to pre-registration nursing curricula. Assessing the student's perceptions of their CLE is essential to adjust clinical placement to trainees' needs. Clinical Learning Environment Inventory (CLEI) appears to be widely used in measuring CLE, but no previous study has reported a full structural validity and its association with students' satisfaction in the Moroccan context. Objectives: This study investigated the psychometric properties of the CLEI and its subscales association with Moroccan nursing students' satisfaction. Methods: The research design was descriptive, cross-sectional, and conducted from March and June 2022 using convenience sampling in three nursing institutes of the Fez-Meknes region of Morocco. The selected sample involved Moroccan nursing students undertaking clinical practice. First, exploratory factor analysis (EFA) was used to determine the factor structure of the pilot sample (N = 143). The second sample (N = 206) was then used to confirm this structure using partial least squares structural equation modeling (PLS-SEM) confirmatory composite analysis (CCA). Finally, using a bootstrapping method, the significance of the structural path was evaluated. Results: The CLEI scale depicted convergent validity (AVE = 0.56 - 0.71), discriminant validity, estimated by the square roots of AVE and bootstrapped HTMT confidence interval, and significant reliability (rhoC = 0.83 - 0.92). Using a bootstrapping approach, structural path significance displayed a substantial association between task orientation and students' satisfaction (ß = 0.29, p <0.001). This ascertains that nurse students need well-planned guidelines from their facilitators in clinical wards. Conclusions: The CLEI instrument revealed adequate psychometric properties and supported its original structure. As a result, the instrument might be used to measure students' perceptions of their CLE. Task orientation appeared to be the most important factor influencing the students' satisfaction in CLE.

Mercurial risk from dental amalgam use in a population of Moroccan dentists: A latent class regression approach.

BACKGROUND: Dentists using dental amalgam are chronically exposed to low doses of elemental mercury. The complex toxico-kinetics of this systemic toxicant results in polymorphic and variable clinical phenotypes. In this context, adapted statistical methods are required to highlight potential adverse effects of occupational mercury exposure on dentists' health. OBJECTIVE: The present study aims to analyze the distribution of self-reported subjective symptoms, commonly associated with chronic mercury poisoning, according to occupational mercury exposure in a population of Moroccan liberal dentists. METHODS: In order to achieve the defined objectives, a three-step latent class regression was fitted. First a latent class analysis was performed to cluster the studied population according to their declared symptoms. Dentists were then classified in the defined latent classes based on their posterior probabilities. Finally, a logistic regression is fitted to identify predictors associated with the latent classes' membership. RESULTS: The final obtained model showed acceptable calibration and discrimination. Its interpretation revealed that the increase of the frequency of amalgam use was associated with significant higher odds of belonging to the high risk latent class. CONCLUSIONS: The present study represents an initial step towards the development of diagnosis model that predict clinical profiles according to occupational mercury exposure.

Preventive planning against mercury over-exposure among Moroccan dentists using multidimensional statistical methods.

BACKGROUND: Mercury used in dental amalgams constitutes a significant source of chronic exposure to this heavy metal among dentists. Thus, the safety of dental amalgam remains a controversial issue despite its long history of use. In Morocco, most studies about dental mercury were mainly focused on the environmental risk related to the management of mercury-contaminated waste. OBJECTIVE: In order to evaluate the occupational exposure to mercury among liberal dentists practicing in two Moroccan regions, a multidimensional statistical approach was used to analyze the collected data. The main objective was to help establishing a targeted prevention plan aiming to reduce the mercury exposure among Moroccan dentists. METHODS: Fifteen variables from 146 dentists were elected for a three-step classification procedure: a multiple correspondence analysis followed by a hierarchical ascendant clustering consolidated by the k-Means algorithm. RESULTS: Three homogenous clusters were identified. The most important one includes 57.5% of the population as well as the majority of the risky factors. The characterization of these clusters allows proposing concise guidelines for a targeted preventive plan. CONCLUSIONS: A real mercurial risk has been observed in the studied population. However, its impact on health as well as the efficiency of simple preventive recommendations remains to be unveiled.

Modeling Subjective Symptoms Related to Micro-Hydrargyrism in a Population of Moroccan Dentists.

BACKGROUND: The ability of mercury to deposit throughout the body and alter a wide range of molecular and cellular pathways results in a polymorphic and complex clinical phenotype with over 250 possible symptoms. However, some of them are recurrently cited as evoking chronic mercury poisoning. In this light, dentists users of dental amalgams are chronically exposed to mercury so that in-depth epidemiological investigations and adapted statistical methods are required to highlight adverse effects of this exposure. METHODS: In order to study the health impact of the occupational mercury exposure in a population of liberal dentists practicing in two Moroccan regions, a list of eighteen subjective symptoms commonly associated with micro-hydrargyrism was drawn up. Then, seven statisctical models adapted to count data were fitted. Finally, three methods were used to compare their relative performance in order to choose the most appropriate one. RESULTS: The adopted logical path, from the response variable selection till models' comparison, led us to lean towards quasi-Poisson regression as the best way to predict the number of symptoms declared by dentists according to mercury exposure. CONCLUSIONS: Interpretation of the selected model allowed us to conclude that the reduction of dental amalgam use allows the reduction of subjective symptoms related to mercury exposure.

Ajuga iva water extract antihypertensive effect on stroke-prone spontaneously hypertensive rats, vasorelaxant effects ex vivo and in vitro activity of fractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Ajuga iva (L.) Schreb. (Labiatae) (AI) is used in folk medicine for a variety of ailments, including diabetes mellitus and hypertension. AIM OF THE STUDY: In this work, we aimed to investigate the antihypertensive and vasorelaxant effects of AI aqueous extract in stroke prone spontaneously hypertensive rats (SHR-SP). MATERIAL AND METHODS: Male SHR-SP rats were orally force-fed AI aqueous extract (500 mg/kg body weight) daily for one week. Systolic blood pressure and urine output were recorded in vivo by non-invasive methods. AI vasoactive effects on noradrenaline contractile response and acetylcholine-evoked relaxation were assessed ex vivo on aorta rings of treated and untreated SHR-SP rats. AI extract was then subjected to bio-guided fractionation using solvents of increasing polarity. For each fraction, in vitro vasorelaxation assay was performed on noradrenaline-precontracted aorta of Wistar rats, in the absence/presence of N-nitro-L-arginine (L-NNA). HPLC analysis of AI total extract, and the most in vitro active AI residual aqueous extract fraction (A1) was performed using naringin, naringenin, apigenin, apigenin 7-O-glucoside as marker compounds. RESULTS: AI aqueous extract (500 mg/kg) significantly (P < 0.05) decreased systolic blood pressure (SBP) in SHR-SP rats, while not affecting the urine output. In ex vivo experiments, the total extract decreased contractile response to noradrenaline of aortic rings isolated from AI-treated SHR-SP rats with or without addition of N-nitro-L-arginine, but endothelium dependent relaxation evoked by acetylcholine in noradrenaline-contracted aortic rings was not affected by the extract treatment. In vitro experiments on AI aqueous extract fractions showed that its polar fraction was the only one affecting in vitro noradrenaline induced contractions, but only in an endothelium dependent manner. This fraction was shown by HPLC-UV to contain flavonoid glycosides among other polar compounds whose activity and mode of action may be modified in vivo by metabolization. CONCLUSION: These results support the use of AI as antihypertensive treatment in folk medicine. The systolic blood pressure decrease may be attributed at least in part to vasorelaxant glycosylated/polar phenolic compounds as flavonoids and/or their metabolites.

Inherited Cardiac Arrhythmia Syndromes: Focus on Molecular Mechanisms Underlying TRPM4 Channelopathies.

The Transient Receptor Potential Melastatin 4 (TRPM4) is a transmembrane N-glycosylated ion channel that belongs to the large family of TRP proteins. It has an equal permeability to Na+ and K+ and is activated via an increase of the intracellular calcium concentration and membrane depolarization. Due to its wide distribution, TRPM4 dysfunction has been linked with several pathophysiological processes, including inherited cardiac arrhythmias. Many pathogenic variants of the TRPM4 gene have been identified in patients with different forms of cardiac disorders such as conduction defects, Brugada syndrome, and congenital long QT syndrome. At the cellular level, these variants induce either gain- or loss-of-function of TRPM4 channels for similar clinical phenotypes. However, the molecular mechanisms associating these functional alterations to the clinical phenotypes remain poorly understood. The main objective of this article is to review the major cardiac TRPM4 channelopathies and recent advances regarding their genetic background and the underlying molecular mechanisms.

[Mercury exposure and dentists' health status in two regions of centrall Morocco: descriptive cross-sectional survey]. / Exposition au mercure et état de santé des médecins dentistes de deux régions du centre du Maroc: enquête transversale descriptive.

INTRODUCTION: mercury exposure is recognized as a worldwide public health concern. However, the effect of long-term exposure to low-doses of this heavy metal is still subject to debate. Due to the use of mercury in dental amalgam, dental health care professionals are chronically exposed to low-doses of this metal. In this context, we have conducted a descriptive cross-sectional survey among liberal dentists in two regions of the center of Morocco. In parallel, the global health status of participants was investigated to assess the relevance of a subsequent etiological survey. METHODS: data were collected through a self-reported questionnaire. Occupational exposure of dentists to mercury was evaluated based on their use of dental amalgam. Moreover, the other common factors increasing the background pollution or inducing exposure peaks were also investigated. On the other hand, smoking, vaccination, fish consumption, and the number of dental amalgam in mouth were considered as non-occupational exposure sources. Finally, the self-reported global health problems of participants were collected. RESULTS: 192 dentists were included in the present study. Seventy-six percent (76.04%) of them declared using dental amalgam in their practice. Moreover, the presence of dental amalgam in mouth was identified as the main non-occupational source of exposure to mercury (63.45% of participants). Finally, most of participants (46.35%) have expressed neuropsychological complaints. CONCLUSION: altogether, our results revealed a real mercury exposure in the studied population. Thus, effective preventive measures should be promoted to minimize the mercury exposure in dental offices. Moreover, an etiological study will be of great interest to reveal the impact of mercury exposure in this population.

Biophysical Characterization of Epigallocatechin-3-Gallate Effect on the Cardiac Sodium Channel Nav1.5.

Epigallocatechin-3-Gallate (EGCG) has been extensively studied for its protective effect against cardiovascular disorders. This effect has been attributed to its action on multiple molecular pathways and transmembrane proteins, including the cardiac Nav1.5 channels, which are inhibited in a dose-dependent manner. However, the molecular mechanism underlying this effect remains to be unveiled. To this aim, we have characterized the EGCG effect on Nav1.5 using electrophysiology and molecular dynamics (MD) simulations. EGCG superfusion induced a dose-dependent inhibition of Nav1.5 expressed in tsA201 cells, negatively shifted the steady-state inactivation curve, slowed the inactivation kinetics, and delayed the recovery from fast inactivation. However, EGCG had no effect on the voltage-dependence of activation and showed little use-dependent block on Nav1.5. Finally, MD simulations suggested that EGCG does not preferentially stay in the center of the bilayer, but that it spontaneously relocates to the membrane headgroup region. Moreover, no sign of spontaneous crossing from one leaflet to the other was observed, indicating a relatively large free energy barrier associated with EGCG transport across the membrane. These results indicate that EGCG may exert its biophysical effect via access to its binding site through the cell membrane or via a bilayer-mediated mechanism.

AFM and FluidFM Technologies: Recent Applications in Molecular and Cellular Biology.

Atomic force microscopy (AFM) is a widely used imaging technique in material sciences. After becoming a standard surface-imaging tool, AFM has been proven to be useful in addressing several biological issues such as the characterization of cell organelles, quantification of DNA-protein interactions, cell adhesion forces, and electromechanical properties of living cells. AFM technique has undergone many successful improvements since its invention, including fluidic force microscopy (FluidFM), which combines conventional AFM with microchanneled cantilevers for local liquid dispensing. This technology permitted to overcome challenges linked to single-cell analyses. Indeed, FluidFM allows isolation and injection of single cells, force-controlled patch clamping of beating cardiac cells, serial weighting of micro-objects, and single-cell extraction for molecular analyses. This work aims to review the recent studies of AFM implementation in molecular and cellular biology.

In Silico Evaluation of the Potential Antiarrhythmic Effect of Epigallocatechin-3-Gallate on Cardiac Channelopathies.

Ion channels are transmembrane proteins that allow the passage of ions according to the direction of their electrochemical gradients. Mutations in more than 30 genes encoding ion channels have been associated with an increasingly wide range of inherited cardiac arrhythmias. In this line, ion channels become one of the most important molecular targets for several classes of drugs, including antiarrhythmics. Nevertheless, antiarrhythmic drugs are usually accompanied by some serious side effects. Thus, developing new approaches could offer added values to prevent and treat the episodes of arrhythmia. In this sense, green tea catechins seem to be a promising alternative because of the significant effect of Epigallocatechin-3-Gallate (E3G) on the electrocardiographic wave forms of guinea pig hearts. Thus, the aim of this study was to evaluate the benefits-risks balance of E3G consumption in the setting of ion channel mutations linked with aberrant cardiac excitability phenotypes. Two gain-of-function mutations, Nav1.5-p.R222Q and Nav1.5-p.I141V, which are linked with cardiac hyperexcitability phenotypes were studied. Computer simulations of action potentials (APs) show that 30 µM E3G reduces and suppresses AP abnormalities characteristics of these phenotypes. These results suggest that E3G may have a beneficial effect in the setting of cardiac sodium channelopathies displaying a hyperexcitability phenotype.

Variants of Transient Receptor Potential Melastatin Member 4 in Childhood Atrioventricular Block.

BACKGROUND: Transient receptor potential melastatin member 4 (TRPM4) is a nonselective cation channel. TRPM4 mutations have been linked to cardiac conduction disease and Brugada syndrome. The mechanisms underlying TRPM4-dependent conduction slowing are not fully understood. The aim of this study was to characterize TRPM4 genetic variants found in patients with congenital or childhood atrioventricular block. METHODS AND RESULTS: Ninety-one patients with congenital or childhood atrioventricular block were screened for candidate genes. Five rare TRPM4 genetic variants were identified and investigated. The variants were expressed heterologously in HEK293 cells. Two of the variants, A432T and A432T/G582S, showed decreased expression of the protein at the cell membrane; inversely, the G582S variant showed increased expression. Further functional characterization of these variants using whole-cell patch-clamp configuration showed a loss of function and a gain of function, respectively. We hypothesized that the observed decrease in expression was caused by a folding and trafficking defect. This was supported by the observation that incubation of these variants at lower temperature partially rescued their expression and function. Previous studies have suggested that altered SUMOylation of TRPM4 may cause a gain of function; however, we did not find any evidence that supports SUMOylation as being directly involved for the gain-of-function variant. CONCLUSIONS: This study underpins the role of TRPM4 in the cardiac conduction system. The loss-of-function variants A432T/G582S found in 2 unrelated patients with atrioventricular block are most likely caused by misfolding-dependent altered trafficking. The ability to rescue this variant with lower temperature may provide a novel use of pharmacological chaperones in treatment strategies.

Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I.

BACKGROUND: Progressive cardiac conduction disease (PCCD) is one of the most common cardiac conduction disturbances. It has been causally related to rare mutations in several genes including SCN5A, SCN1B, TRPM4, LMNA and GJA5. METHODS AND RESULTS: In this study, by applying targeted next-generation sequencing (NGS) in 95 unrelated patients with PCCD, we have identified 13 rare variants in the TRPM4 gene, two of which are currently absent from public databases. This gene encodes a cardiac calcium-activated cationic channel which precise role and importance in cardiac conduction and disease is still debated. One novel variant, TRPM4-p.I376T, is carried by the proband of a large French 4-generation pedigree. Systematic familial screening showed that a total of 13 family members carry the mutation, including 10 out of the 11 tested affected individuals versus only 1 out of the 21 unaffected ones. Functional and biochemical analyses were performed using HEK293 cells, in whole-cell patch-clamp configuration and Western blotting. TRPM4-p.I376T results in an increased current density concomitant to an augmented TRPM4 channel expression at the cell surface. CONCLUSIONS: This study is the first extensive NGS-based screening of TRPM4 coding variants in patients with PCCD. It reports the third largest pedigree diagnosed with isolated Progressive Familial Heart Block type I and confirms that this subtype of PCCD is caused by mutation-induced gain-of-expression and function of the TRPM4 ion channel.

Cellular hyper-excitability caused by mutations that alter the activation process of voltage-gated sodium channels.

Voltage-gated sodium channels (Nav) are widely expressed as macro-molecular complexes in both excitable and non-excitable tissues. In excitable tissues, the upstroke of the action potential is the result of the passage of a large and rapid influx of sodium ions through these channels. NaV dysfunction has been associated with an increasingly wide range of neurological, muscular and cardiac disorders. The purpose of this review is to summarize the recently identified sodium channel mutations that are linked to hyper-excitability phenotypes and associated with the alteration of the activation process of voltage gated sodium channels. Indeed, several clinical manifestations that demonstrate an alteration of tissue excitability were recently shown to be strongly associated with the presence of mutations that affect the activation process of the Nav. These emerging genotype-phenotype correlations have expanded the clinical spectrum of sodium channelopathies to include disorders which feature a hyper-excitability phenotype that may or may not be associated with a cardiomyopathy. The p.I141V mutation in SCN4A and SCN5A, as well as its homologous p.I136V mutation in SCN9A, are interesting examples of mutations that have been linked to inherited hyperexcitability myotonia, exercise-induced polymorphic ventricular arrhythmias and erythromelalgia, respectively. Regardless of which sodium channel isoform is investigated, the substitution of the isoleucine to valine in the locus 141 induces similar modifications in the biophysical properties of the Nav by shifting the voltage-dependence of steady state activation toward more negative potentials.

Force-controlled patch clamp of beating cardiac cells.

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Complex genetic background in a large family with Brugada syndrome.

The Brugada syndrome (BrS) is an inherited arrhythmia characterized by ST-segment elevation in V1-V3 leads and negative T wave on standard ECG. BrS patients are at risk of sudden cardiac death (SCD) due to ventricular tachyarrhythmia. At least 17 genes have been proposed to be linked to BrS, although recent findings suggested a polygenic background. Mutations in SCN5A, the gene coding for the cardiac sodium channel Nav1.5, have been found in 15-30% of index cases. Here, we present the results of clinical, genetic, and expression studies of a large Iranian family with BrS carrying a novel genetic variant (p.P1506S) in SCN5A. By performing whole-cell patch-clamp experiments using HEK293 cells expressing wild-type (WT) or p.P1506S Nav1.5 channels, hyperpolarizing shift of the availability curve, depolarizing shift of the activation curve, and hastening of the fast inactivation process were observed. These mutant-induced alterations lead to a loss of function of Nav1.5 and thus suggest that the p.P1506S variant is pathogenic. In addition, cascade familial screening found a family member with BrS who did not carry the p.P1506S mutation. Additional next generation sequencing analyses revealed the p.R25W mutation in KCNH2 gene in SCN5A-negative BrS patients. These findings illustrate the complex genetic background of BrS found in this family and the possible pathogenic role of a new SCN5A genetic variant.

Functional interaction between S1 and S4 segments in voltage-gated sodium channels revealed by human channelopathies.

The p.I141V mutation of the voltage-gated sodium channel is associated with several clinical hyper-excitability phenotypes. To understand the structural bases of the p.I141V biophysical alterations, molecular dynamics simulations were performed. These simulations predicted that the p.I141V substitution induces the formation of a hydrogen bond between the Y168 residue of the S2 segment and the R225 residue of the S4 segment. We generated a p.I141V-Y168F double mutant for both the Nav1.4 and Nav1.5 channels. The double mutants demonstrated the abolition of the functional effects of the p.I141V mutation, consistent with the formation of a specific interaction between Y168-S2 and R225-S4. The single p.Y168F mutation, however, positively shifted the activation curve, suggesting a compensatory role of these residues on the stability of the voltage-sensing domain.

Complex Brugada syndrome inheritance in a family harbouring compound SCN5A and CACNA1C mutations.

Brugada syndrome (BrS) is characterized by ST-segment elevation in the right precordial leads and is associated with increased risk of sudden cardiac death. We have recently reported families with BrS and SCN5A mutations where some affected members do not carry the familial mutation. We evaluated the involvement of additional genetic determinants for BrS in an affected family. We identified three distinct gene variants within a family presenting BrS (5 individuals), cardiac conduction defects (CCD, 3 individuals) and shortened QT interval (4 individuals). The first mutation is nonsense, p.Q1695*, lying within the SCN5A gene, which encodes for NaV1.5, the α-subunit of the cardiac Na(+) channel. The second mutation is missense, p.N300D, and alters the CACNA1C gene, which encodes the α-subunit CaV1.2 of the L-type cardiac Ca(2+) channel. The SCN5A mutation strictly segregates with CCD. Four out of the 5 BrS patients carry the CACNA1C variant, and three of them present shortened QT interval. One of the BrS patients carries none of these mutations but a rare variant located in the ABCC9 gene as well as his asymptomatic mother. Patch-clamp studies identified a loss-of-function of the mutated CaV1.2 channel. Western-blot experiments showed a global expression defect while increased mobility of CaV1.2 channels on cell surface was revealed by FRAP experiments. Finally, computer simulations of the two mutations recapitulated patient phenotypes. We report a rare CACNA1C mutation as causing BrS and/or shortened QT interval in a family also carrying a SCN5A stop mutation, but which does not segregate with BrS. This study underlies the complexity of BrS inheritance and its pre-symptomatic genetic screening interpretation.

Gain-of-function mutation of the SCN5A gene causes exercise-induced polymorphic ventricular arrhythmias.

BACKGROUND: Over the past 15 years, a myriad of mutations in genes encoding cardiac ion channels and ion channel interacting proteins have been linked to a long list of inherited atrial and ventricular arrhythmias. The purpose of this study was to identify the genetic and functional determinants underlying exercise-induced polymorphic ventricular arrhythmia present in a large multigenerational family. METHODS AND RESULTS: A large 4-generation family presenting with exercise-induced polymorphic ventricular arrhythmia, which was followed for 10 years, was clinically characterized. A novel SCN5A mutation was identified via whole exome sequencing and further functionally evaluated by patch-clamp studies using human embryonic kidney 293 cells. Of 37 living family members, a total of 13 individuals demonstrated ≥50 multiformic premature ventricular complexes or ventricular tachycardia upon exercise stress tests when sinus rate exceeded 99±17 beats per minute. Sudden cardiac arrest occurred in 1 individual during follow-up. Exome sequencing identified a novel missense mutation (p.I141V) in a highly conserved region of the SCN5A gene, encoding the Nav1.5 sodium channel protein that cosegregated with the arrhythmia phenotype. The mutation p.I141V shifted the activation curve toward more negative potentials and increased the window current, whereas action potential simulations suggested that it lowered the excitability threshold of cardiac cells. CONCLUSIONS: Gain-of-function of Nav1.5 may cause familial forms of exercise-induced polymorphic ventricular arrhythmias.

A long QT mutation substitutes cholesterol for phosphatidylinositol-4,5-bisphosphate in KCNQ1 channel regulation.

INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. METHODS AND RESULTS: COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. CONCLUSIONS: We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.