Journal of General Physiology最新文献

{"title":"Epilepsy-associated SCN2A (NaV1.2) variants exhibit diverse and complex functional properties.","authors":"Christopher H Thompson, Franck Potet, Tatiana V Abramova, Jean-Marc DeKeyser, Nora F Ghabra, Carlos G Vanoye, John J Millichap, Alfred L George","doi":"10.1085/jgp.202313375","DOIUrl":"10.1085/jgp.202313375","url":null,"abstract":"<p><p>Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID). Previous work to determine the functional consequences of SCN2A variants yielded a paradigm in which predominantly gain-of-function variants cause neonatal-onset epilepsy, whereas loss-of-function variants are associated with ASD and ID. However, this framework was derived from a limited number of studies conducted under heterogeneous experimental conditions, whereas most disease-associated SCN2A variants have not been functionally annotated. We determined the functional properties of SCN2A variants using automated patch-clamp recording to demonstrate the validity of this method and to examine whether a binary classification of variant dysfunction is evident in a larger cohort studied under uniform conditions. We studied 28 disease-associated variants and 4 common variants using two alternatively spliced isoforms of NaV1.2 expressed in HEK293T cells. Automated patch-clamp recording provided a valid high throughput method to ascertain detailed functional properties of NaV1.2 variants with concordant findings for variants that were previously studied using manual patch clamp. Many epilepsy-associated variants in our study exhibited complex patterns of gain- and loss-of-functions that are difficult to classify by a simple binary scheme. The higher throughput achievable with automated patch clamp enables study of variants with greater standardization of recording conditions, freedom from operator bias, and enhanced experimental rigor. This approach offers an enhanced ability to discern relationships between channel dysfunction and neurodevelopmental disorders.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 10","pages":""},"PeriodicalIF":3.3,"publicationDate":"2023-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10424433/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10516793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The fast block to polyspermy breaks with convention.","authors":"Ben Short","doi":"10.1085/jgp.202313478","DOIUrl":"10.1085/jgp.202313478","url":null,"abstract":"<p><p>JGP study (Komondor et al. 2023. J. Gen. Physiol. https://doi.org/10.1085/jgp.202213258) reveals that conventional PLC activation pathways are not required for the fertilization-induced depolarization of Xenopus eggs that prevents the entry of additional sperm.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2023-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10499036/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10589601","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Urea transport in human red blood cells: Donor variation compared to chloride, glucose, and water transport.","authors":"Jonas Leifelt, Morten Hanefeld Dziegiel, Jesper Brahm","doi":"10.1085/jgp.202213321","DOIUrl":"10.1085/jgp.202213321","url":null,"abstract":"<p><p>We determined the permeability (P, cm/s) of unmodified human red blood cells (HRBC) to urea (Pu), chloride (PCl), glucose (Pglu), and water diffusion (Pd) under conditions of self-exchange (SE) with the continuous flow tube method at pH 7.2, 25°C. Among 24 donors, Pu at 1 mM varied >100%. Two of the donors were also tested in 1983. Their Pu had decreased by 77 and 90%. High age in males and Kidd genotype Jk(a+,b+), but not blood types AB0, appear related to low Pu. For one of the two donors, PCl (150 mM, 38°C, pH 7.2), Pglu (1 mM, 38°C, pH 7.2), and Pd (55.5 M, 25°C, pH 7.2) were determined then and now and showed no significant changes with age. The results from six more donors show donor PCl, Pglu, and Pd in the range of ≈1%. PCl and Pglu are vital for the metabolism of cells and tissues, and we see but little donor variation, and so far, no phenotypes without glucose (GLUT1) and anion (AE1) transporters in HRBC. Phenotypes with no urea transporter (UT-B) or no water transporters (aquaporin, AQP1) are registered and are compatible with life. Our results are in line with the concept that the solutes do not share pathways in common. The great donor variation in Pu must be considered in comparative transport physiological studies.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 10","pages":""},"PeriodicalIF":3.3,"publicationDate":"2023-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10397051/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10040481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs.","authors":"Kayla M Komondor, Rachel E Bainbridge, Katherine G Sharp, Anuradha R Iyer, Joel C Rosenbaum, Anne E Carlson","doi":"10.1085/jgp.202213258","DOIUrl":"10.1085/jgp.202213258","url":null,"abstract":"<p><p>Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+-activated Cl- channel TMEM16A. To do so, fertilization activates phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCβ. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCβ. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2023-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10405425/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10230125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mechanistic understanding of KCNQ1 activating polyunsaturated fatty acid analogs.","authors":"Jessica J Jowais, Samira Yazdi, Alessia Golluscio, Vanessa Olivier-Meo, Sara I Liin, H Peter Larsson","doi":"10.1085/jgp.202313339","DOIUrl":"10.1085/jgp.202313339","url":null,"abstract":"<p><p>The KCNQ1 channel is important for the repolarization phase of the cardiac action potential. Loss of function mutations in KCNQ1 can cause long QT syndrome (LQTS), which can lead to cardiac arrythmia and even sudden cardiac death. We have previously shown that polyunsaturated fatty acids (PUFAs) and PUFA analogs can activate the cardiac KCNQ1 channel, making them potential therapeutics for the treatment of LQTS. PUFAs bind to KCNQ1 at two different binding sites: one at the voltage sensor (Site I) and one at the pore (Site II). PUFA interaction at Site I shifts the voltage dependence of the channel to the left, while interaction at Site II increases maximal conductance. The PUFA analogs, linoleic-glycine and linoleic-tyrosine, are more effective than linoleic acid at Site I, but less effective at Site II. Using both simulations and experiments, we find that the larger head groups of linoleic-glycine and linoleic-tyrosine interact with more residues than the smaller linoleic acid at Site I. We propose that this will stabilize the negatively charged PUFA head group in a position to better interact electrostatically with the positively charges in the voltage sensor. In contrast, the larger head groups of linoleic-glycine and linoleic-tyrosine compared with linoleic acid prevent a close fit of these PUFA analogs in Site II, which is more confined. In addition, we identify several KCNQ1 residues as critical PUFA-analog binding residues, thereby providing molecular models of specific interactions between PUFA analogs and KCNQ1. These interactions will aid in future drug development based on PUFA-KCNQ1 channel interactions.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 10","pages":""},"PeriodicalIF":3.8,"publicationDate":"2023-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10394376/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9941361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Determinants of iFGF13-mediated regulation of myocardial voltage-gated sodium (NaV) channels in mouse.","authors":"Adrien Lesage, Maxime Lorenzini, Sophie Burel, Marine Sarlandie, Floriane Bibault, Cecilia Lindskog, Daniel Maloney, Jonathan R Silva, R Reid Townsend, Jeanne M Nerbonne, Céline Marionneau","doi":"10.1085/jgp.202213293","DOIUrl":"10.1085/jgp.202213293","url":null,"abstract":"<p><p>Posttranslational regulation of cardiac NaV1.5 channels is critical in modulating channel expression and function, yet their regulation by phosphorylation of accessory proteins has gone largely unexplored. Using phosphoproteomic analysis of NaV channel complexes from adult mouse left ventricles, we identified nine phosphorylation sites on intracellular fibroblast growth factor 13 (iFGF13). To explore the potential roles of these phosphosites in regulating cardiac NaV currents, we abolished expression of iFGF13 in neonatal and adult mouse ventricular myocytes and rescued it with wild-type (WT), phosphosilent, or phosphomimetic iFGF13-VY. While the increased rate of closed-state inactivation of NaV channels induced by Fgf13 knockout in adult cardiomyocytes was completely restored by adenoviral-mediated expression of WT iFGF13-VY, only partial rescue was observed in neonatal cardiomyocytes after knockdown. The knockdown of iFGF13 in neonatal ventricular myocytes also shifted the voltage dependence of channel activation toward hyperpolarized potentials, a shift that was not reversed by WT iFGF13-VY expression. Additionally, we found that iFGF13-VY is the predominant isoform in adult ventricular myocytes, whereas both iFGF13-VY and iFGF13-S are expressed comparably in neonatal ventricular myocytes. Similar to WT iFGF13-VY, each of the iFGF13-VY phosphomutants studied restored NaV channel inactivation properties in both models. Lastly, Fgf13 knockout also increased the late Na+ current in adult cardiomyocytes, and this effect was restored with expression of WT and phosphosilent iFGF13-VY. Together, our results demonstrate that iFGF13 is highly phosphorylated and displays differential isoform expression in neonatal and adult ventricular myocytes. While we found no roles for iFGF13 phosphorylation, our results demonstrate differential effects of iFGF13 on neonatal and adult mouse ventricular NaV channels.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 9","pages":""},"PeriodicalIF":3.8,"publicationDate":"2023-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10374952/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9948025","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The structural and functional integrities of porcine myocardium are mostly preserved by cryopreservation.","authors":"Weikang Ma, Kyoung Hwan Lee, Christine E Delligatti, M Therese Davis, Yahan Zheng, Henry Gong, Jonathan A Kirk, Roger Craig, Thomas Irving","doi":"10.1085/jgp.202313345","DOIUrl":"10.1085/jgp.202313345","url":null,"abstract":"<p><p>Structural and functional studies of heart muscle are important to gain insights into the physiological bases of cardiac muscle contraction and the pathological bases of heart disease. While fresh muscle tissue works best for these kinds of studies, this is not always practical to obtain, especially for heart tissue from large animal models and humans. Conversely, tissue banks of frozen human hearts are available and could be a tremendous resource for translational research. It is not well understood, however, how liquid nitrogen freezing and cryostorage may impact the structural integrity of myocardium from large mammals. In this study, we directly compared the structural and functional integrity of never-frozen to previously frozen porcine myocardium to investigate the consequences of freezing and cryostorage. X-ray diffraction measurements from hydrated tissue under near-physiological conditions and electron microscope images from chemically fixed porcine myocardium showed that prior freezing has only minor effects on structural integrity of the muscle. Furthermore, mechanical studies similarly showed no significant differences in contractile capabilities of porcine myocardium with and without freezing and cryostorage. These results demonstrate that liquid nitrogen preservation is a practical approach for structural and functional studies of myocardium.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 9","pages":""},"PeriodicalIF":3.3,"publicationDate":"2023-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10318404/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9761775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"EAAT5 glutamate transporter rapidly binds glutamate with micromolar affinity in mouse rods.","authors":"Wallace B Thoreson, Bhavana Chhunchha","doi":"10.1085/jgp.202313349","DOIUrl":"10.1085/jgp.202313349","url":null,"abstract":"<p><p>Light responses of rod photoreceptor cells in the retina are encoded by changes in synaptic glutamate release that is in turn shaped by reuptake involving EAAT5 plasma membrane glutamate transporters. Heterologously expressed EAAT5 activates too slowly upon glutamate binding to support significant uptake. We tested EAAT5 activation in mouse rods in vivo by stimulating glutamate transporter anion currents (IA(glu)) with UV flash photolysis of MNI-glutamate, varying flash intensity to vary glutamate levels. Responses to uncaging rose rapidly with time constants of 2-3 ms, similar to IA(glu) events arising from spontaneous release. Spontaneous release events and IA(glu) evoked by weak flashes also declined with similar time constants of 40-50 ms. Stronger flashes evoked responses that decayed more slowly. Time constants were twofold faster at 35°C, suggesting that they reflect transporter kinetics, not diffusion. Selective EAAT1 and EAAT2 inhibitors had no significant effect, suggesting IA(glu) in rods arises solely from EAAT5. We calibrated glutamate levels attained during flash photolysis by expressing a fluorescent glutamate sensor iGluSnFr in cultured epithelial cells. We compared fluorescence at different glutamate concentrations to fluorescence evoked by photolytic uncaging of MNI-glutamate. The relationship between flash intensity and glutamate yielded EC50 values for EAAT5 amplitude, decay time, and rise time of ∼10 μM. Micromolar affinity and rapid activation of EAAT5 in rods show it can rapidly bind synaptic glutamate. However, we also found that EAAT5 currents are saturated by the synchronous release of only a few vesicles, suggesting limited capacity and a role for glial uptake at higher release rates.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 9","pages":""},"PeriodicalIF":3.3,"publicationDate":"2023-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10359920/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9899731","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"D-type K+ current rules the function of electrically coupled neurons in a species-specific fashion.","authors":"Antonella Dapino, Federico Davoine, Sebastian Curti","doi":"10.1085/jgp.202313353","DOIUrl":"10.1085/jgp.202313353","url":null,"abstract":"<p><p>Electrical synapses supported by gap junctions are known to form networks of electrically coupled neurons in many regions of the mammalian brain, where they play relevant functional roles. Yet, how electrical coupling supports sophisticated network operations and the contribution of the intrinsic electrophysiological properties of neurons to these operations remain incompletely understood. Here, a comparative analysis of electrically coupled mesencephalic trigeminal (MesV) neurons uncovered remarkable difference in the operation of these networks in highly related species. While spiking of MesV neurons might support the recruitment of coupled cells in rats, this rarely occurs in mice. Using whole-cell recordings, we determined that the higher efficacy in postsynaptic recruitment in rat's MesV neurons does not result from coupling strength of larger magnitude, but instead from the higher excitability of coupled neurons. Consistently, MesV neurons from rats present a lower rheobase, more hyperpolarized threshold, as well as a higher ability to generate repetitive discharges, in comparison to their counterparts from mice. This difference in neuronal excitability results from a significantly higher magnitude of the D-type K+ current (ID) in MesV neurons from mice, indicating that the magnitude of this current gates the recruitment of postsynaptic-coupled neurons. Since MesV neurons are primary afferents critically involved in the organization of orofacial behaviors, activation of a coupled partner could support lateral excitation, which by amplifying sensory inputs may significantly contribute to information processing and the organization of motor outputs.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 9","pages":""},"PeriodicalIF":3.8,"publicationDate":"2023-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10308032/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9934705","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Light drives the developmental progression of outer retinal function.","authors":"Paul J Bonezzi, Matthew J Tarchick, Brittney D Moore, Jordan M Renna","doi":"10.1085/jgp.202213262","DOIUrl":"10.1085/jgp.202213262","url":null,"abstract":"<p><p>The complex nature of rod and cone photoreceptors and the light-evoked responsivity of bipolar cells in the mature rodent retina have been well characterized. However, little is known about the emergent light-evoked response properties of the mouse retina and the role light plays in shaping these emergent responses. We have previously demonstrated that the outer retina is responsive to green light as early as postnatal day 8 (P8). Here, we characterize the progression of both photoreceptors (rods and cones) and bipolar cell responses during development and into adulthood using ex vivo electroretinogram recordings. Our data show that the majority of photoreceptor response at P8 originates from cones and that these outputs drive second-order bipolar cell responses as early as P9. We find that the magnitude of the photoresponse increases concurrently with each passing day of postnatal development and that many functional properties of these responses, as well as the relative rod/cone contributions to the total light-evoked response, are age dependent. We compare these responses at eye opening and maturity to age-matched animals raised in darkness and found that the absence of light diminishes emergent and mature cone-to-bipolar cell signaling. Furthermore, we found cone-evoked responses to be significantly slower in dark-reared retinas. Together, this work characterizes the developmental photoresponsivity of the mouse retina while highlighting the importance of properly timed sensory input for the maturation of the first visual system synapse.</p>","PeriodicalId":54828,"journal":{"name":"Journal of General Physiology","volume":"155 9","pages":""},"PeriodicalIF":3.8,"publicationDate":"2023-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10336150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10171439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}