Improving the Sequence Coverage of Integral Membrane Proteins during Hydrogen/Deuterium Exchange Mass Spectrometry Experiments.

Insight into the structure-function relationship of membrane proteins is important to understand basic cell function and inform drug development, as these are common targets for drugs. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established technique for the study of protein conformational dynamics and has shown compatibility with membrane proteins. However, the digestion and mass analysis of peptides from membrane proteins can be challenging, severely limiting the HDX-MS experiment. Here we compare the digestion of four integral membrane proteins-Cl-/H+ exchange transporter (ClC-ec1), leucine transporter (LeuT), dopamine transporter (DAT), and serotonin transporter (SERT)-by the use of porcine pepsin and three alternative aspartic proteases either in-solution or immobilized on-column in an optimized HDX-MS-compatible workflow. Pepsin was the most favorable for the digestion of ClC-ec1 and LeuT, providing coverage of 82.2 and 33.2% of the respective protein sequence; however, the alternative proteases surpassed pepsin for the digestion of DAT and SERT. By also screening quench solution additives, we observe that the denaturant urea was beneficial, resulting in improved sequence coverage of all membrane proteins, in contrast to guanidine hydrochloride. Furthermore, significant improvements in sequence coverage were achieved by tailoring the chromatography to handle hydrophobic peptides. Overall, we demonstrate that the susceptibility of membrane proteins to proteolytic digestion during HDX-MS is highly protein-specific. Our results highlight the importance of having multiple proteases and different quench buffer additives in the HDX-MS toolbox and the need to carefully screen a range of digestion conditions to successfully optimize the HDX-MS analysis of integral membrane proteins.

TBX5 variants and cardiac phenotype: A systematic review of the literature and a novel variant.

T-Box Transcription Factor 5 (TBX5) variants are associated with Holt-Oram syndrome. Holt-Oram syndrome display phenotypic variability, regarding upper limb defects, congenital heart defects, and arrhythmias. To investigate the genotype-phenotype relationship between TBX5 variants and cardiac disease, we performed a systematic review of the literature. Through the systematic review we identified 108 variants in TBX5 associated with a cardiac phenotype in 277 patients. Arrhythmias were more frequent in patients with a missense variant (48% vs 30%, p = 0.009) and upper limb abnormalities were more frequent in patients with protein-truncating variants (85% vs 64%, p = 0.0008). We found clustering of missense variants in the T-box domain. Furthermore, we present a family with atrial septal defects. By whole exome sequencing, we identified a novel missense variant p.Phe232Leu in TBX5. The cardiac phenotype included atrial septal defect, arrhythmias, heart failure, and dilated cardiomyopathy. Clinical examination revealed subtle upper limb abnormalities. Thus, the family corresponds to the diagnostic criteria of Holt-Oram syndrome. We provide an overview of cardiac phenotypes associated with TBX5 variants and show an increased risk of arrhythmias associated to missense variants compared to protein-truncating variants. We report a novel missense variant in TBX5 in a family with an atypical Holt-Oram syndrome phenotype.

Exercise capacity in the Danish Fontan population remains stable after ten years of follow-up - is physical activity the key to success?

BACKGROUND: Most Fontan patients have impaired exercise capacity, and a further decline in exercise capacity over time seems inevitable. However, few longitudinal studies exist, and there is a lack of data from newer eras. We aimed to describe the natural evolution of exercise capacity over a 10-year period in a contemporary, population-based cohort of Danish Fontan patients. METHODS: The study was a nationwide, prospective study. A cardiopulmonary exercise test (CPET) was used to assess the exercise capacity. All Danish Fontan patients who participated in a national study in 2011 (CPET1), were invited to a follow-up visit in 2021 (CPET2). All patients who completed CPET1 and CPET2 with a respiratory exchange ratio over 1.0 were included. The main outcome was percent predicted VO2peak (%pred VO2peak). At the time of CPET2, patients filled out a questionnaire including questions regarding physical activity. RESULTS: Seventy-seven patients completed both CPET1 and CPET2, and seventy patients completed the questionnaire. The time interval between the two CPETs was 10.4 ± 0.9 years. The median age was 15 years at CPET1 and 26 years at CPET2. The exercise capacity remained stable with a mean %pred VO2peak of 53.8 ± 11.3 at CPET1 and 55.6 ± 10.9 in CPET2 (p = 0.314). Higher levels of vigorous physical activity were associated with higher %pred VO2peak in CPET2 in a multivariate regression model. CONCLUSION: The %pred VO2peak remained stable over a ten-year period in this population-based Fontan cohort. Higher levels of self-reported vigorous physical activity were associated with higher %pred VO2peak in the most recent CPET.

Comparison of Outcome in Patients With Familial Versus Spontaneous Atrial Septal Defect.

Patients with atrial septal defects (ASDs) have increased mortality and morbidity. This can only partly be explained by hemodynamic changes caused by the ASD, suggesting additional underlying causes. Patients with an ASD have an increased burden of pathogenic gene variants in ASD-related genes, indicating genetics as an important factor in etiology. Inheritance of genetic variants with high impact can cause ASD in relatives (familial ASD). This study aimed to investigate whether lifelong outcomes were different in patients with familial ASD compared with patients with sporadic ASD. We used health registries and a nationwide cohort of 2,151 patients with ASD to compare the incidences of atrial fibrillation or flutter (together abbreviated as AF), heart failure, and mortality between patients with familial and sporadic ASD using Cox proportional hazard ratio and Fine and Gray analysis. Patients with familial ASD experienced AF and heart failure earlier in life than patients with sporadic ASD, with hazard ratios of 1.6 and 1.7, respectively. Subdistribution hazard ratios showed an increased risk of AF and heart failure in patients with familial ASD compared with patients with sporadic ASDs (2.3 and 3.1, respectively). Our results suggest that genetic variants with high impact may influence the outcomes of patients with ASD. In conclusion, patients with familial ASD have an increased risk and an earlier onset of AF and heart failure compared with patients with sporadic ASD, hence clinical awareness of arrhythmias and heart failure in patients with familial ASD may lead to timely treatment.

The dopamine transporter antiports potassium to increase the uptake of dopamine.

The dopamine transporter facilitates dopamine reuptake from the extracellular space to terminate neurotransmission. The transporter belongs to the neurotransmitter:sodium symporter family, which includes transporters for serotonin, norepinephrine, and GABA that utilize the Na+ gradient to drive the uptake of substrate. Decades ago, it was shown that the serotonin transporter also antiports K+, but investigations of K+-coupled transport in other neurotransmitter:sodium symporters have been inconclusive. Here, we show that ligand binding to the Drosophila- and human dopamine transporters are inhibited by K+, and the conformational dynamics of the Drosophila dopamine transporter in K+ are divergent from the apo- and Na+-states. Furthermore, we find that K+ increases dopamine uptake by the Drosophila dopamine transporter in liposomes, and visualize Na+ and K+ fluxes in single proteoliposomes using fluorescent ion indicators. Our results expand on the fundamentals of dopamine transport and prompt a reevaluation of the impact of K+ on other transporters in this pharmacologically important family.

Partial Anomalous Pulmonary Venous Connection: Forty-Six Years of Follow-Up.

BACKGROUND: Surgical repair of partial anomalous pulmonary venous connection (PAPVC) may disturb the electrical conduction in the atria. This study documents long-term outcomes, including the late occurrence of atrial tachyarrhythmia and bradyarrhythmia. METHODS: This retrospective study covers all PAPVC operations at Aarhus University Hospital between 1970 and 2010. Outcome measures were arrhythmias, sinus node disease, pacemaker implantation, pathway stenosis (pulmonary vein(s), intra-atrial pathway, and/or superior vena cava), and mortality. Data were collected from databases, surgical protocols, and hospital records until May 2018. RESULTS: A total of 83 patients were included with a postoperative follow-up period up to 46 years. Average age at follow-up was 43 ± 21 years. During follow-up, new-onset atrial fibrillation or atrial flutter appeared in four patients (5%). Sinus node disease was present in nine patients (11%). A permanent pacemaker was implanted in seven patients (8%) at an average of 12.7 years after surgery. Pulmonary venous and/or superior vena cava obstruction was seen in five patients (6%). Stenosis was most prevalent in the two-patch technique, and arrhythmia was most prevalent in the single-patch technique. Sixty-seven (81%) of 83 patients had neither bradyarrhythmias nor tachyarrhythmias or pacemaker need. CONCLUSIONS: This study contributes important long-term data concerning the course of patients who have undergone repair of PAPVC. It confirms that PAPVC can be operated with low postoperative morbidity. However, late-onset stenosis, bradyarrhythmias and tachyarrhythmias, and need for pacemaker call for continued follow-up.

Conformational dynamics of the human serotonin transporter during substrate and drug binding.

The serotonin transporter (SERT), a member of the neurotransmitter:sodium symporter family, is responsible for termination of serotonergic signaling by re-uptake of serotonin (5-HT) into the presynaptic neuron. Its key role in synaptic transmission makes it a major drug target, e.g. for the treatment of depression, anxiety and post-traumatic stress. Here, we apply hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs. Our results reveal significant changes in dynamics in regions TM1, EL3, EL4, and TM12 upon binding co-transported ions (Na+/K+) and ligand-mediated changes in TM1, EL3 and EL4 upon binding 5-HT, the drugs S-citalopram, cocaine and ibogaine. Our results provide a comprehensive direct view of the conformational response of SERT upon binding both biologically relevant substrate/ions and ligands of pharmaceutical interest, thus advancing our understanding of the structure-function relationship in SERT.

Substrate-induced conformational dynamics of the dopamine transporter.

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.