The Journal of General Physiology最新文献

{"title":"Membrane potential and Ca2+ concentration dependence on pressure and vasoactive agents in arterial smooth muscle: A model.","authors":"Arthur Karlin","doi":"10.1085/jgp.201511380","DOIUrl":"https://doi.org/10.1085/jgp.201511380","url":null,"abstract":"<p><p>Arterial smooth muscle (SM) cells respond autonomously to changes in intravascular pressure, adjusting tension to maintain vessel diameter. The values of membrane potential (Vm) and sarcoplasmic Ca(2+) concentration (Ca(in)) within minutes of a change in pressure are the results of two opposing pathways, both of which use Ca(2+) as a signal. This works because the two Ca(2+)-signaling pathways are confined to distinct microdomains in which the Ca(2+) concentrations needed to activate key channels are transiently higher than Ca(in). A mathematical model of an isolated arterial SM cell is presented that incorporates the two types of microdomains. The first type consists of junctions between cisternae of the peripheral sarcoplasmic reticulum (SR), containing ryanodine receptors (RyRs), and the sarcolemma, containing voltage- and Ca(2+)-activated K(+) (BK) channels. These junctional microdomains promote hyperpolarization, reduced Ca(in), and relaxation. The second type is postulated to form around stretch-activated nonspecific cation channels and neighboring Ca(2+)-activated Cl(-) channels, and promotes the opposite (depolarization, increased Ca(in), and contraction). The model includes three additional compartments: the sarcoplasm, the central SR lumen, and the peripheral SR lumen. It incorporates 37 protein components. In addition to pressure, the model accommodates inputs of α- and β-adrenergic agonists, ATP, 11,12-epoxyeicosatrienoic acid, and nitric oxide (NO). The parameters of the equations were adjusted to obtain a close fit to reported Vm and Ca(in) as functions of pressure, which have been determined in cerebral arteries. The simulations were insensitive to ± 10% changes in most of the parameters. The model also simulated the effects of inhibiting RyR, BK, or voltage-activated Ca(2+) channels on Vm and Ca(in). Deletion of BK β1 subunits is known to increase arterial-SM tension. In the model, deletion of β1 raised Ca(in) at all pressures, and these increases were reversed by NO.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"79-96"},"PeriodicalIF":3.8,"publicationDate":"2015-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201511380","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33428512","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":"A marginal band of microtubules transports and organizes mitochondria in retinal bipolar synaptic terminals.","authors":"Malkolm Graffe,&nbsp;David Zenisek,&nbsp;Justin W Taraska","doi":"10.1085/jgp.201511396","DOIUrl":"https://doi.org/10.1085/jgp.201511396","url":null,"abstract":"<p><p>A set of bipolar cells in the retina of goldfish contains giant synaptic terminals that can be over 10 µm in diameter. Hundreds of thousands of synaptic vesicles fill these terminals and engage in continuous rounds of exocytosis. How the cytoskeleton and other organelles in these neurons are organized to control synaptic activity is unknown. Here, we used 3-D fluorescence and 3-D electron microscopy to visualize the complex subcellular architecture of these terminals. We discovered a thick band of microtubules that emerged from the axon to loop around the terminal periphery throughout the presynaptic space. This previously unknown microtubule structure associated with a substantial population of mitochondria in the synaptic terminal. Drugs that inhibit microtubule-based kinesin motors led to accumulation of mitochondria in the axon. We conclude that this prominent microtubule band is crucial to the transport and localization of mitochondria into the presynaptic space to provide the sustained energy necessary for continuous transmitter release in these giant synaptic terminals. </p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"109-17"},"PeriodicalIF":3.8,"publicationDate":"2015-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201511396","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33431092","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":"Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel.","authors":"Doreen Badheka,&nbsp;Istvan Borbiro,&nbsp;Tibor Rohacs","doi":"10.1085/jgp.201411336","DOIUrl":"https://doi.org/10.1085/jgp.201411336","url":null,"abstract":"<p><p>Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5'-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily. </p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"65-77"},"PeriodicalIF":3.8,"publicationDate":"2015-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201411336","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33428511","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":"Friends of Physiology: an interview with Clara Franzini-Armstrong and Clay Armstrong.","authors":"Elizabeth M Adler","doi":"10.1085/jgp.201311115","DOIUrl":"https://doi.org/10.1085/jgp.201311115","url":null,"abstract":"In early September, I took a trip to Woods Hole, where Incoming Editor-in-Chief Sharona Gordon and I attended the 67th Annual Meeting of the Society of General Physiologists. This year’s meeting saw the unveiling of the Society’s Friends of Physiology Lecture Series Honoring Clara Franzini-","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"479"},"PeriodicalIF":3.8,"publicationDate":"2013-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201311115","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40270054","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":"Structural basis of ion permeation gating in Slo2.1 K+ channels.","authors":"Priyanka Garg,&nbsp;Alison Gardner,&nbsp;Vivek Garg,&nbsp;Michael C Sanguinetti","doi":"10.1085/jgp.201311064","DOIUrl":"https://doi.org/10.1085/jgp.201311064","url":null,"abstract":"The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K+ channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K+ channel that is activated by intracellular Na+ and Cl−. Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation.","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"523-42"},"PeriodicalIF":3.8,"publicationDate":"2013-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201311064","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40272218","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":"Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel.","authors":"Roland S Wu,&nbsp;Guoxia Liu,&nbsp;Sergey I Zakharov,&nbsp;Neelesh Chudasama,&nbsp;Howard Motoike,&nbsp;Arthur Karlin,&nbsp;Steven O Marx","doi":"10.1085/jgp.201210891","DOIUrl":"https://doi.org/10.1085/jgp.201210891","url":null,"abstract":"<p><p>Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2-β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2-β4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other β2-β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"105-17"},"PeriodicalIF":3.8,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201210891","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40202043","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":"Effect of muscle length on cross-bridge kinetics in intact cardiac trabeculae at body temperature.","authors":"Nima Milani-Nejad,&nbsp;Ying Xu,&nbsp;Jonathan P Davis,&nbsp;Kenneth S Campbell,&nbsp;Paul M L Janssen","doi":"10.1085/jgp.201210894","DOIUrl":"https://doi.org/10.1085/jgp.201210894","url":null,"abstract":"<p><p>Dynamic force generation in cardiac muscle, which determines cardiac pumping activity, depends on both the number of sarcomeric cross-bridges and on their cycling kinetics. The Frank-Starling mechanism dictates that cardiac force development increases with increasing cardiac muscle length (corresponding to increased ventricular volume). It is, however, unclear to what extent this increase in cardiac muscle length affects the rate of cross-bridge cycling. Previous studies using permeabilized cardiac preparations, sub-physiological temperatures, or both have obtained conflicting results. Here, we developed a protocol that allowed us to reliably and reproducibly measure the rate of tension redevelopment (k(tr); which depends on the rate of cross-bridge cycling) in intact trabeculae at body temperature. Using K(+) contractures to induce a tonic level of force, we showed the k(tr) was slower in rabbit muscle (which contains predominantly β myosin) than in rat muscle (which contains predominantly α myosin). Analyses of k(tr) in rat muscle at optimal length (L(opt)) and 90% of optimal length (L(90)) revealed that k(tr) was significantly slower at L(opt) (27.7 ± 3.3 and 27.8 ± 3.0 s(-1) in duplicate analyses) than at L(90) (45.1 ± 7.6 and 47.5 ± 9.2 s(-1)). We therefore show that k(tr) can be measured in intact rat and rabbit cardiac trabeculae, and that the k(tr) decreases when muscles are stretched to their optimal length under near-physiological conditions, indicating that the Frank-Starling mechanism not only increases force but also affects cross-bridge cycling kinetics.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"133-9"},"PeriodicalIF":3.8,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201210894","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40202045","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":"Induced pluripotent stem cells used to reveal drug actions in a long QT syndrome family with complex genetics.","authors":"Cecile Terrenoire,&nbsp;Kai Wang,&nbsp;Kelvin W Chan Tung,&nbsp;Wendy K Chung,&nbsp;Robert H Pass,&nbsp;Jonathan T Lu,&nbsp;Jyh-Chang Jean,&nbsp;Amel Omari,&nbsp;Kevin J Sampson,&nbsp;Darrell N Kotton,&nbsp;Gordon Keller,&nbsp;Robert S Kass","doi":"10.1085/jgp.201210899","DOIUrl":"https://doi.org/10.1085/jgp.201210899","url":null,"abstract":"<p><p>Understanding the basis for differential responses to drug therapies remains a challenge despite advances in genetics and genomics. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to investigate the pharmacology of disease processes in therapeutically and genetically relevant primary cell types in vitro and to interweave clinical and basic molecular data. We report here the derivation of iPSCs from a long QT syndrome patient with complex genetics. The proband was found to have a de novo SCN5A LQT-3 mutation (F1473C) and a polymorphism (K897T) in KCNH2, the gene for LQT-2. Analysis of the biophysics and molecular pharmacology of ion channels expressed in cardiomyocytes (CMs) differentiated from these iPSCs (iPSC-CMs) demonstrates a primary LQT-3 (Na(+) channel) defect responsible for the arrhythmias not influenced by the KCNH2 polymorphism. The F1473C mutation occurs in the channel inactivation gate and enhances late Na(+) channel current (I(NaL)) that is carried by channels that fail to inactivate completely and conduct increased inward current during prolonged depolarization, resulting in delayed repolarization, a prolonged QT interval, and increased risk of fatal arrhythmia. We find a very pronounced rate dependence of I(NaL) such that increasing the pacing rate markedly reduces I(NaL) and, in addition, increases its inhibition by the Na(+) channel blocker mexiletine. These rate-dependent properties and drug interactions, unique to the proband's iPSC-CMs, correlate with improved management of arrhythmias in the patient and provide support for this approach in developing patient-specific clinical regimens.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"61-72"},"PeriodicalIF":3.8,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201210899","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40202040","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":"Eschewing ischemia or responding to it.","authors":"Elizabeth M Adler","doi":"10.1085/jgp.201210947","DOIUrl":"https://doi.org/10.1085/jgp.201210947","url":null,"abstract":"","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"1-2"},"PeriodicalIF":3.8,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201210947","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40202039","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":"Ablation of cardiac myosin-binding protein-C accelerates contractile kinetics in engineered cardiac tissue.","authors":"Willem J de Lange,&nbsp;Adrian C Grimes,&nbsp;Laura F Hegge,&nbsp;J Carter Ralphe","doi":"10.1085/jgp.201210837","DOIUrl":"https://doi.org/10.1085/jgp.201210837","url":null,"abstract":"<p><p>Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin-binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C-null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca(2+) transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C-null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca(2+) transient kinetics. Strikingly, the ability of cMyBP-C-null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca(2+) transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.</p>","PeriodicalId":173753,"journal":{"name":"The Journal of General Physiology","volume":" ","pages":"73-84"},"PeriodicalIF":3.8,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1085/jgp.201210837","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40202041","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}