Voltage-gated NaV channels are modulated by two different noncovalent accessory subunits: β1 and β3. Zhu et al. present data showing that β1 and β3 cause distinct effects on channel gating because they interact with NaV channels at different locations. β3 regulates the voltage sensor in domain III, whereas β1 regulates the one in domain IV.

Background Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na+ current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the four (DI-DIV) VSDs to define molecular mechanisms of NaV1.5 modulation. Method and Results Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry (VCF). Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of two Brugada Syndrome (BrS) mutations (A735V and G752R). A735V shifted DII-VSD voltage-dependence to depolarized potentials, while G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, even though DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate ofINa activation and myocyte excitability. Conclusions Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate its activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal BrS mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications and anti-arrhythmic drugs alter NaV1.5 at the molecular level.

Hsu et al. probe voltage-gated Na+ channels that are inactivation deficient with voltage-clamp fluorometry. They find that in the time domain of an action potential, the voltage-sensing domain (VSD) of domain IV regulates fast inactivation onset while the domain III VSD determines its recovery.

Late-breaking abstract presentation during the plenary session at the ISPD conference, Tuesday, 14 July 2015.Funding sources: This work was funded by Life Codexx AG. Conflicts of interest: WH is an employee and shareholder of LifeCodexx AG. SG, US and JB are employees of LifeCodexx AG. CvK is a member of the Supervisory Board of LifeCodexx AG and shareholder of LifeCodexx AG. The other authors declare no conflict of interest.Non-invasive prenatal testing (NIPT) for common fetal trisomies has been radically changing prenatal diagnosis.1 Several hundreds of thousands of tests are being performed worldwide every year. Until today the fetal fraction in cell-free maternal plasma DNA is the most critical determinant for a successful NIPT analysis which is affected by certain parameters such as maternal weight 2 or the presence of an aneuploidy. 3 No other pregnancy-related factors, crucial for a reliable NIPT analysis, have been identified yet. We provide the first report describing an effect of drugs on circulating plasma nucleic acids and subsequent NIPT results. Low molecular weight heparin (LMWH) is widely used as anticoagulants in placenta-mediated pregnancy complication. 4 We show that in pregnant women on LMWH medication the cell-free plasma DNA contains a higher proportion of small DNA fragments featuring an unusually high guanosin-cytosin (GC) content than in unaffected women. This apparently biases NIPT results so that they cannot be interpreted correctly and even may provide false results. For such cases our report provides guidance that blood sampling for NIPT should ideally occur right before the next application of LMWH when the LMWH level is lowest in blood. Within three months of the year 2014, we performed a total of 1614 routine NIPT analyses (Method in the Supplementary Appendix). In 12 samples (0.74%), results could not be interpreted correctly due to an elevated GC content of higher than 44.0% in comparison to the average GC content of 42.0% estimated for the majority of analyzed plasma samples (data not shown). In correlation with the elevated GC content the z-score of chromosome 18 increased whereas the z-scores for chromosome 13 and chromosome 21 decreased. Thus, not assessing the GC content of a sample as a quality criterion of NIPT leads to falsepositive results for trisomy 18 or false negative results for trisomy 13 or trisomy 21. Repeat analysis on the routine backup blood samples (taken at the same time of the first blood sample) failed for the same reason. The clinical records of the patients with failed analyzed samples revealed that nine of these twelve women were on LMWH prophylaxis. The indications for the LMWH medication were thrombosis prophylaxis, protein C deficiency, risk of pulmonary embolism and repeated miscarriages. For five of these women (Table 1 and Table S1 in the Supplementary Appendix), an additional blood sampling was carried out right before the next pending LMWH injection, when the plasma level from the previous injection was lowest. Then, routine NIPT of these pre-LM...