The UCSF COMET Consortium, Michael Matthay, David J. E rl e , P re scot t G. Woodruff, Charles Langelier, Kirsten K an ge la ri s, C ar ol yn M.

Certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid PI(3,5)P2. Purified Vo complexes bind preferentially to this lipid, and the cytosolic domain of one Vo subunit shows PI(3,5)P2-dependent recruitment to membranes in vivo. Lipid interactions with V-ATPases could provide compartment-specific regulation.

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.

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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.

The immunogenicity of subunit vaccines can be augmented by formulating them into nanoparticles. We conjugated recombinant trimetric influenza A/Aichi/2/68(H3N2) hemagglutinin (HA) onto functionalized gold nanoparticle (AuNP) surfaces in a repetitive, oriented configuration. To further improve the immunogenicity, we generated Toll-like receptor 5 (TLR5) agonist flagellin (FliC)-coupled AuNPs as particulate adjuvants. Intranasal immunizations with an AuNP-HA and AuNP-FliC particle mixture elicited strong mucosal and systemic immune responses that protected hosts against lethal influenza challenges. Compared with the AuNP-HA alone group, the addition of AuNP-FliC improved mucosal B cell responses as characterized by elevated influenza specific IgA and IgG levels in nasal, tracheal, and lung washes. AuNP-HA/AuNP-FliC also stimulated antigen-specific interferon-γ (IFN-γ)-secreting CD4 cell proliferation and induced strong effector CD8 T cell activation. Our results indicate that intranasal co-delivery of antigen and adjuvant-displaying AuNPs enhanced vaccine efficacy by inducing potent cellular immune responses.

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.

Rationale: Mutations in the SCN5A gene, encoding the α subunit of the Nav1.5 channel, cause a life-threatening form of cardiac arrhythmia, long QT syndrome type 3 (LQT3). Mexiletine, which is structurally related to the Na + channel-blocking anesthetic lidocaine, is used to treat LQT3 patients. However, the patient response is variable, depending on the genetic mutation in SCN5A . Objective: The goal of this study is to understand the molecular basis of patients’ variable responses and build a predictive statistical model that can be used to personalize mexiletine treatment based on patient’s genetic variant. Methods and Results: We monitored the cardiac Na + channel voltage-sensing domain (VSD) conformational dynamics simultaneously with other gating properties for the LQT3 variants. To systematically identify the relationship between mexiletine block and channel biophysical properties, we used a system-based statistical modeling approach to connect the multivariate properties to patient phenotype. We found that mexiletine altered the conformation of the Domain III VSD, which is the same VSD that many tested LQT3 mutations affect. Analysis of 15 LQT3 variants showed a strong correlation between the activation of the Domain III-VSD and the strength of the inhibition of the channel by mexiletine. Based on this improved molecular-level understanding, we generated a systems-based model based on a dataset of 32 LQT3 patients, which then successfully predicted the response of 7 out of 8 patients to mexiletine in a blinded, retrospective trial. Conclusions: Our results imply that the modulated receptor theory of local anesthetic action, which confines local anesthetic binding effects to the channel pore, should be revised to include drug interaction with the Domain III-VSD. Using an algorithm that incorporates this mode of action, we can predict patient-specific responses to mexiletine, improving therapeutic decision making.