Voltage-gated sodium channels are critical for the generation and propagation of electrical signals in most excitable cells. Activation of Na+ channels initiates an action potential, and fast inactivation facilitates repolarization of the membrane by the outward K+ current. Fast inactivation is also the main determinant of the refractory period between successive electrical impulses. Although the voltage sensor of domain IV (DIV) has been implicated in fast inactivation, it remains unclear whether the activation of DIV alone is sufficient for fast inactivation to occur. Here, we functionally neutralize each specific voltage sensor by mutating several critical arginines in the S4 segment to glutamines. We assess the individual role of each voltage-sensing domain in the voltage dependence and kinetics of fast inactivation upon its specific inhibition. We show that movement of the DIV voltage sensor is the rate-limiting step for both development and recovery from fast inactivation. Our data suggest that activation of the DIV voltage sensor alone is sufficient for fast inactivation to occur, and that activation of DIV before channel opening is the molecular mechanism for closed-state inactivation. We propose a kinetic model of sodium channel gating that can account for our major findings over a wide voltage range by postulating that DIV movement is both necessary and sufficient for fast inactivation.

Voltage-dependent Na+ channels are crucial for electrical signalling in excitable cells. Membrane depolarization initiates asynchronous movements in four non-identical voltage-sensing domains of the Na+ channel. It remains unclear to what extent this structural asymmetry influences pore gating as compared with outwardly rectifying K+ channels, where channel opening results from a final concerted transition of symmetric pore gates. Here we combine single channel recordings, cysteine accessibility and voltage clamp fluorimetry to probe the relationships between voltage sensors and pore conformations in an inactivation deficient Nav1.4 channel. We observe three distinct conductance levels such that DI-III voltage sensor activation is kinetically correlated with formation of a fully open pore, whereas DIV voltage sensor movement underlies formation of a distinct subconducting pore conformation preceding inactivation in wild-type channels. Our experiments reveal that pore gating in sodium channels involves multiple transitions driven by asynchronous movements of voltage sensors. These findings shed new light on the mechanism of coupling between activation and fast inactivation in voltage-gated sodium channels.

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltagesensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels. electrophysiology | Nav1.4 | outer pore conformation

Rationale While much progress has been made in the resolution of the cellular hierarchy underlying cardiogenesis, our understanding of chamber-specific myocardium differentiation remains incomplete. Objective To better understand ventricular myocardium differentiation, we targeted the ventricle-specific gene, Irx4, in mouse embryonic stem cells to generate a reporter cell line. Methods and Results Using an antibiotic-selection approach, we purified Irx4+ cells in vitro from differentiating embryoid bodies. The isolated Irx4+ cells proved to be highly proliferative and presented Cxcr4, Pdgfr-alpha, Flk1 and Flt1 on the cell surface. Single Irx4+ ventricular progenitor cells (VPC) exhibited cardiovascular potency, generating endothelial cells, smooth muscle cells and ventricular myocytes in vitro. The ventricular specificity of the Irx4+ population was further demonstrated in vivo as VPCs injected into the cardiac crescent subsequently produced Mlc2v+ myocytes that exclusively contributed to the nascent ventricle at E9.5. These findings support the existence of a newly identified ventricular myocardial progenitor. Conclusions This is the first report of a multipotent cardiac progenitor that contributes progeny specific to the ventricular myocardium.

molecular basis for how these chromatin enzymes recognize the 200 kDa nucleosome is largely unknown. My laboratory investigates how chromatin enzymes interact with the nucleosome. We determined the first crystal structure of a chromatin protein in complex with the nucleosome. Our structure of the 300 kDa RCC1/nucleosome core particle complex at 2.9 Å resolution explains how RCC1's b-propeller domain recognizes the architecture of the nucleosome through a combination of both protein-protein and protein-DNA interactions. Our crystal structure also provides a first atomic view of the nucleosome core particle containing the Widom 601 nucleosome positioning sequence. We find that the 601 DNA forms a 145 bp nucleosome core particle and is thus overwound compared to the human alpha-satellite DNA used in prior nucleosome crystals. I will also discuss the molecular basis for why the Widom 601 DNA sequence is a strong nucleosome positioning sequence.

Our original study (1) used visual inspection as well as biological and geochemical analyses of corals and the surrounding sediment to provide complementary and compelling evidence linking the Deepwater Horizon (DWH) oil spill to the presence of damaged deepwater corals and brittle stars 11 km from the site of the leaking oil.The probability that the impact to this coral community occurred independently of the DWH spill can be estimated on the basis of three facts. (i) This is the only site among 20 deep-water coral communities associated with authigenic seep carbonates in the northern Gulf of Mexico where visual inspection over the past decade has revealed evidence of notable damage to corals.(ii) The presence of dead and dying tissue and the attachment of living ophiuroids to the corals indicate that the impact was recent ( Fig. 1). (iii) The average age of four coral colonies sampled from the site is 460 ± 35 y [according to radiocarbon dating as in Prouty et al. (2)]. Assuming that an independent event had an equal chance of occurring at any of the other seep-related coral sites (1 in 20) and during any of the past 460 y at this site (1 in 460) yields a probability of the damage to corals happening coincidently at this place and time of approximately 0.0001.In addition, there is no evidence from Bureau of Ocean and Energy Management seismic data, National Oceanic and Atmospheric Administration multibeam data, or high-resolution autonomous underwater vehicle multibeam data to indicate slope failure and the underwater landslide suggested by Boehm and Carragher (3) as an alternate explanation for the damage to this site. It is also noteworthy that the coral community examined in our study (1) is on top of a discrete ridge. There is no known mechanism by which material from an underwater landslide would gather at the top of a ridge and not also be apparent in the surrounding area.The coral community examined in our original article is 11 km to the southwest of the Macondo well at a depth of 1,370 m (1), placing it in the path of a documented deep-water plume enriched with petroleum hydrocarbons. A maximum of oil constituents centered at ∼1,400 m was observed within 2 km of these corals between June 23 and 27, 2010 (4), and levels of polycyclic aromatic hydrocarbons considered to be toxic to marine organisms were measured up to a distance of 13 km to the southwest of the Macondo well, at depths between 1,000 and 1,400 m between May 9 and 16, 2010 (5). Both of these studies support our findings (1) and describe discrete measurements that represent a minimum amount of petroleum hydrocarbons that could have impacted coral communities over the 86 d of the DWH spill.The consistent biomarker ratios between coral samples and the oil from the DWH spill were determined using comprehensive 2D gas chromatography coupled to a time-of-flight mass spectrometer (GC×GC-TOF-MS). Although oil samples in the area are indeed difficult to distinguish, GC×GC-TOF-MS is capable of separating, identifying, and quantifying compounds that ...

Inherited erythromelalgia (IEM), characterized by episodic burning pain and erythema of the extremities, is produced by gain-of-function mutations in sodium channel Na v 1.7, which is preferentially expressed in nociceptive and sympathetic neurons. Most patients do not respond to pharmacotherapy, although a few patients have been reported as showing partial relief with lidocaine or mexiletine. We report here a new IEM Na v 1.7 mutation and its favorable response to mexiletine. SCN9A exons from the proband were amplified and sequenced. Whole-cell patch-clamp analysis was used to characterize wild-type and mutant Na v 1.7 channels in mammalian cells. A 7-year-old girl, with a two-year history of symmetric burning pain and erythema in her hands and feet, was diagnosed with erythromelalgia. Treatment with mexiletine reduced the number and severity of pain episodes. We identified a single nucleotide substitution (T2616G) in exon 15, not present in 200 ethnically-matched control alleles, which substitutes valine 872 by glycine (V872G) within DII/S5. V872G shifts activation by -10 mV, slows deactivation, and generates larger ramp currents. We observed a stronger use-dependent inhibition by mexiletine of V872G compared to wild-type channels. The Na V 1.7/V872G mutation provides a molecular basis for DRG hyperexcitability which can produce pain. While most IEM patients do not respond to pharmacotherapy, this patient displayed a favorable response to mexiletine, which appears to be due to use-dependent block of mutant channels. Continued relief from pain, even after mexiletine was discontinued in this patient, might suggest that early treatment may slow the progression of the disease.

sensor movement may underlie the destabilization of the fast-inactivated state in NaV1.5. To test this, we expressed NaV1.5 channels in Xenopus oocytes and recorded gating currents using a cut-open voltage clamp with extracellular solution titrated to either pH 7.4 (control) or pH 6.0. At pH 6.0, compared to pH 7.4, the V1/2 of the Q(V) curve was significantly depolarized (from À57.854.3 mV to À40.855.1 mV). Additionally, the slow time constant of charge recovery was significantly reduced from 16.155.0 ms at pH 7.4 to 9.754.2 ms at pH 6.0. These data suggest a molecular basis for the increased persistent and window currents previously shown in NaV1.5 channels at reduced extracellular pH. Specifically, protons may electrostatically affect the rate of voltage sensor movement, either by directly binding to extracellular residues (e.g. H880) or indirectly by binding to carboxylates in the pore domain (Kahn et al., J Physiol. 2002, 543).