Passenger and heavy vehicle collisions with pedestrians: Assessment of injury mechanisms and risk.

OBJECTIVE: Automated Driving System (ADS) fleets are currently being deployed in several dense-urban operational design domains within the United States. In these dense-urban areas, pedestrians have historically comprised a significant portion, and sometimes the majority, of injury and fatal collisions. An expanded understanding of the injury risk in collision events involving pedestrians and human-driven vehicles can inform continued ADS development and safety benefits evaluation. There is no current systematic investigation of United States pedestrian collisions, so this study used reconstruction data from the German In-Depth Accident Study (GIDAS) to develop mechanistic injury risk models for pedestrians involved in collisions with vehicles. DATA SOURCE: The study queried the GIDAS database for cases from 1999 to 2021 involving passenger vehicle or heavy vehicle collisions with pedestrians. METHODS: We describe the injury patterns and frequencies for passenger vehicle-to-pedestrian and heavy vehicle-to-pedestrian collisions, where heavy vehicles included heavy trucks and buses. Injury risk functions were developed at the AIS2+, 3+, 4+ and 5+ levels for pedestrians involved in frontal collisions with passenger vehicles and separately for frontal collisions with heavy vehicles. Model predictors included mechanistic factors of collision speed, pedestrian age, sex, pedestrian height relative to vehicle bumper height, and vehicle acceleration before impact. Children (≤17 y.o.) and elderly (≥65 y.o.) pedestrians were included. We further conducted weighted and imputed analyses to understand the effects of missing data elements and of weighting towards the overall population of German pedestrian crashes. RESULTS: We identified 3,112 pedestrians involved in collisions with passenger vehicles, where 2,524 of those collisions were frontal vehicle strikes. Furthermore, we determined 154 pedestrians involved in collisions with heavy vehicles, where 87 of those identified collisions were frontal vehicle strikes. Children were found to be at higher risk of injury compared to young adults, and the highest risk of serious injuries (AIS 3+) existed for the oldest pedestrians in the dataset. Collisions with heavy vehicles were more likely to produce serious (AIS 3+) injuries at low speeds than collisions with passenger vehicles. Injury mechanisms differed between collisions with passenger vehicles and with heavy vehicles. The initial engagement caused 36% of pedestrians' most-severe injuries in passenger vehicle collisions, compared with 23% in heavy vehicles collisions. Conversely, the vehicle underside caused 6% of the most-severe injuries in passenger vehicle collisions and 20% in heavy vehicles collisions. SIGNIFICANCE: U.S. pedestrian fatalities have risen 59% since their recent recorded low in 2009. It is imperative that we understand and describe injury risk so that we can target effective strategies for injury and fatality reduction. This study builds on previous analyses by including the most modern vehicles, including children and elderly pedestrians, incorporating additional mechanistic predictors, broadening the scope of included crashes, and using multiple imputation and weighting to better estimate these effects relative to the entire population of German pedestrian collisions. This study is the first to investigate the risk of injury to pedestrians in collisions with heavy vehicles based on field data.

Mechanisms of noncovalent ß subunit regulation of NaV channel gating.

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound ß1 and ß3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the ß subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI-DIV), each with a VSD. Our results show that ß1 regulates NaV1.5 by modulating the DIV-VSD, whereas ß3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the ß3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to ß3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that ß3 binds proximally to the DIII-VSD. Molecular-level differences in ß1 and ß3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains.

Functional eukaryotic voltage-gated Na+ (NaV) channels comprise four domains (DI-DIV), each containing six membrane-spanning segments (S1-S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1-S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII-DIV linker, the DIII S4-S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIV-VSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII-DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery.

Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.

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 4 (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. 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 2 Brugada syndrome mutations (A735V and G752R). A735V shifted DII-VSD voltage dependence to depolarized potentials, whereas G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, although 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 of INa activation and myocyte excitability. CONCLUSIONS: Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate channel activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal Brugada syndrome mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications, and antiarrhythmic drugs alter NaV1.5 at the molecular level.

The Xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.