Lawrence, L. | Search bioRxiv

bioRxiv2025

Carboxyl Terminal Domain Missense Mutations Alter Distinct Gating Properties of the Cardiac Sodium Channel

Voltage-gated sodium channels undergo reversible voltage/time-dependent transitions from closed to open and inactivated states. The voltage setpoints and efficiency of cardiac sodium channel Nav1.5 state transitions are crucial for tuning the initiation and conduction of myocardial action potentials. The channels cytoplasmic carboxyl-terminal domain (CTD) regulates gating by intramolecular interactions and by serving as a hub for the binding of accessory proteins. We have investigated the roles of the CTD in intrinsic and FGF homologous factor (FHF)-modulated Nav1.5 gating through structure-guided CTD subdomain mutagenesis. The EF-hand module within the CTD was found to exert the most profound effects on channel gating, strongly influencing voltage-dependence of inactivation and activation, accelerating inactivation from the closed state, decelerating inactivation from the open state, minimizing persistent sodium current, and serving as the binding domain for FHF proteins. Nav1.5D1788K bearing a missense mutation in the EF-hand motif displayed a depolarizing shift in voltage dependence of activation and generated greatly enhanced persistent sodium current without altering the voltage dependence of channel inactivation. Reciprocally, Nav1.5L1861A bearing a different missense mutation in the EF-hand underwent closed-state inactivation at more negative membrane potential and at an accelerated rate, but did not display other phenotypes associated with CTD deletion. Nav1.5V1776A/T1778A bearing mutations in the juxtamembrane region between the EF-hand and the channel pore helices displayed wild-type intrinsic gating properties, while FHF modulation of inactivation gating was impaired. Our channel physiology studies together with prior structural data suggest that the voltage and rate of channel inactivation from the closed state are governed by an intramolecular hydrophobic interaction of the CTD EF-hand with the cytoplasmic inactivation loop helix and the extension of this binding interface upon FHF-induced restructuring of the juxtamembrane region, while a distinct CTD intramolecular electrostatic interaction modulates voltage-dependent activation and minimizes persistent sodium current.

physiology↗

bioRxiv2024

Murine modeling of menstruation identifies immune correlates of protection during Chlamydia muridarum challenge.

The menstrual cycle influences the risk of acquiring sexually transmitted infections (STIs), including Chlamydia trachomatis (C. trachomatis), although the underlying immune contributions are poorly defined. A mouse model simulating the immune-mediated process of menstruation could provide valuable insights into tissue-specific determinants of protection against chlamydial infection within the cervicovaginal and uterine mucosae comprising the female reproductive tract (FRT). Here, we used the pseudopregnancy approach in naive C57Bl/6 mice and performed vaginal challenge with Chlamydia muridarum (C. muridarum) at decidualization, endometrial tissue remodeling, or uterine repair. This strategy identified that the time frame comprising uterine repair correlated with robust infection and greater bacterial burden as compared with mice on hormonal contraception, while challenges during endometrial remodeling were least likely to result in a productive infection. By comparing the infection site at early time points following chlamydial challenge, we found that a greater abundance of innate effector populations and proinflammatory signaling, including IFN{psi} correlated with protection. FRT immune profiling in uninfected mice over pseudopregnancy or in pig-tailed macaques over the menstrual cycle identified NK cell infiltration into the cervicovaginal tissues and lumen over the course of endometrial remodeling. Notably, NK cell depletion over this time frame reversed protection, with mice now productively infected with C. muridarum following challenge. This study shows that the pseudopregnancy murine menstruation model recapitulates immune changes in the FRT as a result of endometrial remodeling and identifies NK cell localization at the FRT as essential for immune protection against primary C. muridarum infection. Author SummaryAlthough the vast majority of women and adolescent girls of reproductive age experience menstruation, we have little insight into how this tissue remodeling process alters mucosal immune defenses against infection by genitourinary pathogens. In this study, we used a murine model of menstruation to investigate how endometrial shedding and repair alters the immune landscape in the female reproductive tract (FRT) to influence chlamydial infections. Using this approach, we identified that endometrial remodeling regulates a substantial pro-inflammatory immune response, including NK cell recruitment into the cervicovaginal tissues, and we further confirmed this phenomenon is occurring in a naturally menstruating species. The localization of NK cells in the FRT at the time of challenge was determined to be responsible for rapid immune protection that reduced C. muridarum burden, as experimental depletion of these cells over this timeframe now led to productive infections. Taken together, this study identifies that murine models of menstruation can be a valuable tool for investigating how the menstrual cycle modulates immune homeostasis and for identifying ways to strengthen mucosal immune defenses against genitourinary pathogens in women.

immunology↗