Interplay between VSD, pore, and membrane lipids in electromechanical coupling in HCN channels

Hyperpolarized-activated and cyclic nucleotide-gated (HCN) channels are the only members of the voltage-gated ion channel superfamily in mammals that open upon hyperpolarization, conferring them pacemaker properties that are instrumental for rhythmic firing of cardiac and neuronal cells. Activation of their voltage-sensor domains (VSD) upon hyperpolarization occurs through a downward movement of the S4 helix bearing the gating charges, which triggers a break in the alpha-helical hydrogen bonding pattern at the level of a conserved Serine residue. Previous structural and molecular simulation studies had however failed to capture pore opening that should be triggered by VSD activation, presumably because of a low VSD/pore electromechanical coupling efficiency and the limited timescales accessible to such techniques. Here, we have used advanced modeling strategies, including enhanced sampling molecular dynamics simulations exploiting comparisons between non-domain swapped voltage-gated ion channel structures trapped in closed and open states to trigger pore gating and characterize electromechanical coupling in HCN1. We propose that the coupling mechanism involves the reorganization of the interfaces between the VSD helices, in particular S4, and the pore-forming helices S5 and S6, subtly shifting the balance between hydrophobic and hydrophilic interactions in a \u27domino effect\u27 during activation and gating in this region. Remarkably, our simulations reveal state-dependent occupancy of lipid molecules at this emergent coupling interface, suggesting a key role of lipids in hyperpolarization-dependent gating. Our model provides a rationale for previous observations and a possible mechanism for regulation of HCN channels by the lipidic components of the membrane

A new splicing isoform of Cacna2d4 mimicking the effects of c.2451insC mutation in the retina: Novel molecular and electrophysiological insights

PURPOSE. Mutations in CACNA2D4 exon 25 cause photoreceptor dysfunction in humans (c.2406CA mutation) and mice (c.2451insC mutation). We investigated the feasibility of an exon-skipping therapeutic approach by evaluating the splicing patterns and functional role of targeted exons. METHODS. Splicing of the targeted a2d4 (CACNA2D4) exons in presence and absence of the mutation was assessed by RT-PCR in vivo on mouse retinae and in vitro in HEK293T cells using splicing-reporter minigenes. Whole-cell patch-clamp recordings were performed to evaluate the impact of different Cacna2d4 variants on the biophysical properties of Cav1.4 Ltype calcium channels (CACNA1F). RESULTS. Splicing analysis revealed the presence of a previously unknown splicing isoform of a2d4 in the retina that truncates the gene open reading frame (ORF) in a similar way as the c.2451insC mutation. This isoform originates from alternative splicing of exon 25 (E25) with a new exon (E25b). Moreover, the c.2451insC mutation has an effect on splicing and increases the proportion of transcripts including E25b. Our electrophysiological analyses showed that only full-length a2d4 was able to increase Cav1.4/b3-mediated currents while all other a2d4 variants did not mediate such effect. CONCLUSIONS. The designed exon-skipping strategy is not applicable because the resulting skipped a2d4 are nonfunctional. a2d4 E25b splicing variant is normally present in mouse retina and mimics the effect of c.2451insC mutation. Since this variant does not promote significant Cav1.4-mediated calcium current, it could possibly mediate a different function, unrelated to modulation of calcium channel properties at the photoreceptor terminals

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Jugendteam — Partizipationsmöglichkeit Jugendlicher in öffentlichen Bibliotheken

Peer Reviewe

Cardiorespiratory Effects of One-Legged High-Intensity Interval Training in Normoxia and Hypoxia: A Pilot Study

A higher-than-average maximal oxygen consumption (VO2max), is closely associated with decreased morbidity and mortality and improved quality of life and acts as a marker of cardiorespiratory fitness. Although there is no consensus about an optimal training method to enhance VO2max, nevertheless training of small muscle groups and repeated exposure to hypoxia seem to be promising approaches. Therefore, this study was aimed at gaining innovative insights into the effects of small muscle group training in normoxia and hypoxia. Thirteen healthy participants were randomly assigned to the hypoxic (HG, n = 7) or normoxic (NG, n = 6) training group. Both groups completed nine high-intensity interval training sessions in 3 wks. The NG performed the training in normoxia (FiO2: 0.21; ~ 600 m) and the HG in hypoxia (FiO2: 0.126; ~ 4500 m). Each session consisted of 4 x 4 min one-legged cycling at 90% of maximal heart rate separated by 4 min recovery periods. Before and after the intervention period, VO2max and peak power output (Wmax) and responses to submaximal cycling (100 and 150 watts) were assessed in a laboratory cycling test. Peak power output significantly improved within both groups (9.6 ± 4.8% and 12.6 ± 8.9% for HG and NG, respectively) with no significant interaction (p = 0.277). However, VO2max only significantly increased after training in hypoxia from 45.4 ± 10.1 to 50.0 ± 9.8 ml/min/kg (10.8 ± 6.0%; p = 0.002) with no significant interaction (p = 0.146). The maximal O2-pulse improved within the HG and demonstrated a significant interaction (p = 0.040). One-legged cycling training significantly improved VO2max and peak power output. Training under hypoxic conditions may generate greater effects on VO2max than a similar training in normoxia and is considered as a promising training method for improving cardiorespiratory fitness

Kinetic Models of Secondary Active Transporters

Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate transport occurs in a closed loop of partial reactions (i.e., a transport cycle). Kinetic models can help to find realistic estimates for conformational transitions between individual states of the transport cycle. When constrained by experimental results, kinetic models can faithfully describe the function of a candidate transporter at a pre-steady state. In addition, we show that kinetic models can accurately predict the intra- and extracellular substrate concentrations maintained by the transporter at a steady state, even under the premise of loose coupling between the electrochemical gradient of the driving ion and of the substrate. We define the criteria for the design of a credible kinetic model of the SLC transporter. Parsimony is the guiding principle of kinetic modeling. We argue, however, that the level of acceptable parsimony is limited by the need to account for the substrate gradient established by a secondary active transporter, and for random order binding of co-substrates and substrate. Random order binding has consistently been observed in transporters of the SLC group

Effects of Regular Long-Term Circuit Training (Once per Week) on Cardiorespiratory Fitness in Previously Sedentary Adults

The purpose of the study was (1) to investigate the effects of regular long-term circuit training (once per week) on cardiorespiratory fitness (CRF) in sedentary adults and (2) to compare training progress with the effects of continued exercise participation by regularly active age-matched individuals. Ten sedentary, middle-aged (51 ± 6 years) individuals (sedentary group, SG) of both sexes performed 32 weeks (1 training session/week) of supervised circuit training and 10 weeks of self-managed training. Effects were compared to an age-matched group (51 ± 8 years; n = 10) of regularly active individuals (active group, AG). CRF (expressed as peak oxygen uptake: VO2peak; peak power output: PPO) and systemic blood pressure (BP) during the incremental test were measured at the start and after the training intervention. CRF decreased significantly within the AG (VO2peak: 43.1 ± 7.3 vs. 40.3 ± 6.5 mL/min/kg, p < 0.05; PPO: 3.3 ± 0.6 vs. 3.1 ± 0.6; p < 0.05) but was maintained in the SG. In addition, significant improvements in restoration of the oxygen level in leg muscles after exercise and reduced systolic BP (180 ± 14 vs. 170 ± 17 mmHg, p = 0.01) at submaximal exercise were found within the SG. However, differences in changes from pre to post did not reach significance between groups. In contrast to the regularly active individuals, circuit training once per week over 32 weeks prevented the aging-related decline of CRF in previously sedentary subjects and reduced systolic BP during submaximal exercise, indicating improved exercise tolerance

eLife / A label-free approach to detect ligand binding to cell surface proteins in real time

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a long-range conformational change. The Gouy-Chapman model predicts that adsorption of charged ligands to surface proteins must produce displacement currents and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.(VLID)468876