The Journal of General Physiology最新文献

{"title":"A flexible GAS belt responds to pore mutations changing the ion selectivity of proton-gated channels.","authors":"Zhuyuan Chen,&nbsp;Sheng Lin,&nbsp;Tianze Xie,&nbsp;Jin-Ming Lin,&nbsp;Cecilia M Canessa","doi":"10.1085/jgp.202112978","DOIUrl":"https://doi.org/10.1085/jgp.202112978","url":null,"abstract":"<p><p>Proton-gated ion channels conduct mainly Na+ to induce postsynaptic membrane depolarization. Finding the determinants of ion selectivity requires knowledge of the pore structure in the open conformation, but such information is not yet available. Here, the open conformation of the hASIC1a channel was computationally modeled, and functional effects of pore mutations were analyzed in light of the predicted structures. The open pore structure shows two constrictions of similar diameter formed by the backbone of the GAS belt and, right beneath it, by the side chains of H28 from the reentrant loop. Models of nonselective mutant channels, but not those that maintain ion selectivity, predict enlargement of the GAS belt, suggesting that this motif is quite flexible and that the loss of stabilizing interactions in the central pore leads to changes in size/shape of the belt. Our results are consistent with the \"close-fit\" mechanism governing selectivity of hASIC1a, wherein the backbone of the GAS substitutes at least part of the hydration shell of a permeant ion to enable crossing the pore constriction.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2022-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594623/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39867791","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Loss of crossbridge inhibition drives pathological cardiac hypertrophy in patients harboring the TPM1 E192K mutation.","authors":"Lorenzo R Sewanan,&nbsp;Jinkyu Park,&nbsp;Michael J Rynkiewicz,&nbsp;Alice W Racca,&nbsp;Nikolaos Papoutsidakis,&nbsp;Jonas Schwan,&nbsp;Daniel L Jacoby,&nbsp;Jeffrey R Moore,&nbsp;William Lehman,&nbsp;Yibing Qyang,&nbsp;Stuart G Campbell","doi":"10.1085/jgp.202012640","DOIUrl":"https://doi.org/10.1085/jgp.202012640","url":null,"abstract":"<p><p>Hypertrophic cardiomyopathy (HCM) is an inherited disorder caused primarily by mutations to thick and thinfilament proteins. Although thin filament mutations are less prevalent than their oft-studied thick filament counterparts, they are frequently associated with severe patient phenotypes and can offer important insight into fundamental disease mechanisms. We have performed a detailed study of tropomyosin (TPM1) E192K, a variant of uncertain significance associated with HCM. Molecular dynamics revealed that E192K results in a more flexible TPM1 molecule, which could affect its ability to regulate crossbridges. In vitro motility assays of regulated actin filaments containing TPM1 E192K showed an overall loss of Ca2+ sensitivity. To understand these effects, we used multiscale computational models that suggested a subtle phenotype in which E192K leads to an inability to completely inhibit actin-myosin crossbridge activity at low Ca2+. To assess the physiological impact of the mutation, we generated patient-derived engineered heart tissues expressing E192K. These tissues showed disease features similar to those of the patients, including cellular hypertrophy, hypercontractility, and diastolic dysfunction. We hypothesized that excess residual crossbridge activity could be triggering cellular hypertrophy, even if the overall Ca2+ sensitivity was reduced by E192K. To test this hypothesis, the cardiac myosin-specific inhibitor mavacamten was applied to patient-derived engineered heart tissues for 4 d followed by 24 h of washout. Chronic mavacamten treatment abolished contractile differences between control and TPM1 E192K engineered heart tissues and reversed hypertrophy in cardiomyocytes. These results suggest that the TPM1 E192K mutation triggers cardiomyocyte hypertrophy by permitting excess residual crossbridge activity. These studies also provide direct evidence that myosin inhibition by mavacamten can counteract the hypertrophic effects of mutant tropomyosin.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/10/66/JGP_202012640.PMC8321830.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39230068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conformations of voltage-sensing domain III differentially define NaV channel closed- and open-state inactivation.","authors":"Paweorn Angsutararux,&nbsp;Po Wei Kang,&nbsp;Wandi Zhu,&nbsp;Jonathan R Silva","doi":"10.1085/jgp.202112891","DOIUrl":"https://doi.org/10.1085/jgp.202112891","url":null,"abstract":"<p><p>Voltage-gated Na+ (NaV) channels underlie the initiation and propagation of action potentials (APs). Rapid inactivation after NaV channel opening, known as open-state inactivation, plays a critical role in limiting the AP duration. However, NaV channel inactivation can also occur before opening, namely closed-state inactivation, to tune the cellular excitability. The voltage-sensing domain (VSD) within repeat IV (VSD-IV) of the pseudotetrameric NaV channel α-subunit is known to be a critical regulator of NaV channel inactivation. Yet, the two processes of open- and closed-state inactivation predominate at different voltage ranges and feature distinct kinetics. How inactivation occurs over these different ranges to give rise to the complexity of NaV channel dynamics is unclear. Past functional studies and recent cryo-electron microscopy structures, however, reveal significant inactivation regulation from other NaV channel components. In this Hypothesis paper, we propose that the VSD of NaV repeat III (VSD-III), together with VSD-IV, orchestrates the inactivation-state occupancy of NaV channels by modulating the affinity of the intracellular binding site of the IFMT motif on the III-IV linker. We review and outline substantial evidence that VSD-III activates in two distinct steps, with the intermediate and fully activated conformation regulating closed- and open-state inactivation state occupancy by altering the formation and affinity of the IFMT crevice. A role of VSD-III in determining inactivation-state occupancy and recovery from inactivation suggests a regulatory mechanism for the state-dependent block by small-molecule anti-arrhythmic and anesthetic therapies.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348240/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39274801","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"State-dependent inhibition of BK channels by the opioid agonist loperamide.","authors":"Alexandre G Vouga,&nbsp;Michael E Rockman,&nbsp;Jiusheng Yan,&nbsp;Marlene A Jacobson,&nbsp;Brad S Rothberg","doi":"10.1085/jgp.202012834","DOIUrl":"https://doi.org/10.1085/jgp.202012834","url":null,"abstract":"<p><p>Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a9/82/JGP_202012834.PMC8352719.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39282696","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HIFs: New arginine mimic inhibitors of the Hv1 channel with improved VSD-ligand interactions.","authors":"Chang Zhao,&nbsp;Liang Hong,&nbsp;Jason D Galpin,&nbsp;Saleh Riahi,&nbsp;Victoria T Lim,&nbsp;Parker D Webster,&nbsp;Douglas J Tobias,&nbsp;Christopher A Ahern,&nbsp;Francesco Tombola","doi":"10.1085/jgp.202012832","DOIUrl":"https://doi.org/10.1085/jgp.202012832","url":null,"abstract":"<p><p>The human voltage-gated proton channel Hv1 is a drug target for cancer, ischemic stroke, and neuroinflammation. It resides on the plasma membrane and endocytic compartments of a variety of cell types, where it mediates outward proton movement and regulates the activity of NOX enzymes. Its voltage-sensing domain (VSD) contains a gated and proton-selective conduction pathway, which can be blocked by aromatic guanidine derivatives such as 2-guanidinobenzimidazole (2GBI). Mutation of Hv1 residue F150 to alanine (F150A) was previously found to increase 2GBI apparent binding affinity more than two orders of magnitude. Here, we explore the contribution of aromatic interactions between the inhibitor and the channel in the presence and absence of the F150A mutation, using a combination of electrophysiological recordings, classic mutagenesis, and site-specific incorporation of fluorinated phenylalanines via nonsense suppression methodology. Our data suggest that the increase in apparent binding affinity is due to a rearrangement of the binding site allowed by the smaller residue at position 150. We used this information to design new arginine mimics with improved affinity for the nonrearranged binding site of the wild-type channel. The new compounds, named \"Hv1 Inhibitor Flexibles\" (HIFs), consist of two \"prongs,\" an aminoimidazole ring, and an aromatic group connected by extended flexible linkers. Some HIF compounds display inhibitory properties that are superior to those of 2GBI, thus providing a promising scaffold for further development of high-affinity Hv1 inhibitors.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/b1/ef/JGP_202012832.PMC8263924.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39158454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"L-type channel inactivation balances the increased peak calcium current due to absence of Rad in cardiomyocytes.","authors":"Brooke M Ahern,&nbsp;Andrea Sebastian,&nbsp;Bryana M Levitan,&nbsp;Jensen Goh,&nbsp;Douglas A Andres,&nbsp;Jonathan Satin","doi":"10.1085/jgp.202012854","DOIUrl":"https://doi.org/10.1085/jgp.202012854","url":null,"abstract":"<p><p>The L-type Ca2+ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (ICa,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates ICa,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of ICa,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak ICa,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in ICa,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell ICa,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of ICa,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β1,β2-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β1,β2-AR still yielded modulated ICa,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca2+ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β1,β2-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/50/cc/JGP_202012854.PMC8289690.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39190100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The number of Z-repeats and super-repeats in nebulin greatly varies across vertebrates and scales with animal size.","authors":"Jochen Gohlke,&nbsp;Paola Tonino,&nbsp;Johan Lindqvist,&nbsp;John E Smith,&nbsp;Henk Granzier","doi":"10.1085/jgp.202012783","DOIUrl":"https://doi.org/10.1085/jgp.202012783","url":null,"abstract":"<p><p>Nebulin is a skeletal muscle protein that associates with the sarcomeric thin filaments and has functions in regulating the length of the thin filament and the structure of the Z-disk. Here we investigated the nebulin gene in 53 species of birds, fish, amphibians, reptiles, and mammals. In all species, nebulin has a similar domain composition that mostly consists of ∼30-residue modules (or simple repeats), each containing an actin-binding site. All species have a large region where simple repeats are organized into seven-module super-repeats, each containing a tropomyosin binding site. The number of super-repeats shows high interspecies variation, ranging from 21 (zebrafish, hummingbird) to 31 (camel, chimpanzee), and, importantly, scales with body size. The higher number of super-repeats in large animals was shown to increase thin filament length, which is expected to increase the sarcomere length for optimal force production, increase the energy efficiency of isometric force production, and lower the shortening velocity of muscle. It has been known since the work of A.V. Hill in 1950 that as species increase in size, the shortening velocity of their muscle is reduced, and the present work shows that nebulin contributes to the mechanistic basis. Finally, we analyzed the differentially spliced simple repeats in nebulin's C terminus, whose inclusion correlates with the width of the Z-disk. The number of Z-repeats greatly varies (from 5 to 18) and correlates with the number of super-repeats. We propose that the resulting increase in the width of the Z-disk in large animals increases the number of contacts between nebulin and structural Z-disk proteins when the Z-disk is stressed for long durations.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/65/cf/JGP_202012783.PMC7754682.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38729577","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Toward an understanding of myofibrillar function in health and disease.","authors":"Richard L Moss,&nbsp;Christine Cremo,&nbsp;Henk L Granzier","doi":"10.1085/jgp.202112880","DOIUrl":"https://doi.org/10.1085/jgp.202112880","url":null,"abstract":"The March 2021 issue of JGP is a collection of peer-reviewed articles focused on the function and dynamic regulation of contractile systems in muscle and non-muscle cells.","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/af/44/JGP_202112880.PMC7905996.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25396297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Convergent evolutionary pathways toward energy saving in muscle?","authors":"Massimo Reconditi","doi":"10.1085/jgp.202012818","DOIUrl":"https://doi.org/10.1085/jgp.202012818","url":null,"abstract":"Reconditi reviews research into the role temperature plays on motor disorders.","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/36/25/JGP_202012818.PMC7879489.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25352789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phosphate has dual roles in cross-bridge kinetics in rabbit psoas single myofibrils.","authors":"Masataka Kawai,&nbsp;Robert Stehle,&nbsp;Gabriele Pfitzer,&nbsp;Bogdan Iorga","doi":"10.1085/jgp.202012755","DOIUrl":"https://doi.org/10.1085/jgp.202012755","url":null,"abstract":"<p><p>In this study, we aimed to study the role of inorganic phosphate (Pi) in the production of oscillatory work and cross-bridge (CB) kinetics of striated muscle. We applied small-amplitude sinusoidal length oscillations to rabbit psoas single myofibrils and muscle fibers, and the resulting force responses were analyzed during maximal Ca2+ activation (pCa 4.65) at 15°C. Three exponential processes, A, B, and C, were identified from the tension transients, which were studied as functions of Pi concentration ([Pi]). In myofibrils, we found that process C, corresponding to phase 2 of step analysis during isometric contraction, is almost a perfect single exponential function compared with skinned fibers, which exhibit distributed rate constants, as described previously. The [Pi] dependence of the apparent rate constants 2πb and 2πc, and that of isometric tension, was studied to characterize the force generation and Pi release steps in the CB cycle, as well as the inhibitory effect of Pi. In contrast to skinned fibers, Pi does not accumulate in the core of myofibrils, allowing sinusoidal analysis to be performed nearly at [Pi] = 0. Process B disappeared as [Pi] approached 0 mM in myofibrils, indicating the significance of the role of Pi rebinding to CBs in the production of oscillatory work (process B). Our results also suggest that Pi competitively inhibits ATP binding to CBs, with an inhibitory dissociation constant of ∼2.6 mM. Finally, we found that the sinusoidal waveform of tension is mostly distorted by second harmonics and that this distortion is closely correlated with production of oscillatory work, indicating that the mechanism of generating force is intrinsically nonlinear. A nonlinear force generation mechanism suggests that the length-dependent intrinsic rate constant is asymmetric upon stretch and release and that there may be a ratchet mechanism involved in the CB cycle.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":""},"PeriodicalIF":3.8,"publicationDate":"2021-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d0/1e/JGP_202012755.PMC7885270.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25379854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}