Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+]cyt) increase as a second messenger. The downstream PM Ca2+channels remain enigmatic. Here, the Extracellular ATP‐induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+]cytwere measured with aequorin, and root transcriptional changes were determined by quantitative real‐time PCR. Two Extracellular ATP‐induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+dependent, requiring CNGC2 but not CNGC4 (its channel co‐subunit in immunity signalling). Activation of PM Ca2+influx currents also required CNGC2. The eATP‐induced [Ca2+]cytincrease and transcriptional response in CYCLIC NUCLEOTIDE‐GATED CHANNEL2 is required for eATP‐induced epidermal Ca2+influx, causing depolarization leading to [Ca2+]cytincrease and damage‐related transcriptional response.
Association of plasma membrane BKCachannels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCachannel (mitoBKCa) by BK‐β subunits is not established. MitoBKCa‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCadisplay properties similar to that of plasma membrane BKCawhen associated with BK‐β1 (left‐shifted voltage dependence of activation, In BK‐β1 knockout mice, cardiac mitoBKCadisplayed a low Co‐expression of BKCawith the BK‐β1 subunit in HeLa cells doubled the density of BKCain mitochondria. The present study supports the view that the cardiac mitoBKCachannel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBKCashapes mitochondrial Ca2+handling.
Association of the plasma membrane BKCachannel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa) with regulatory subunits is unknown. We report that mitoBKCafunctionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBKCais a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K+of 300 pS. Additionally, mitoBKCadisplays a high open probability (
- PAR ID:
- 10456567
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Journal of Physiology
- Volume:
- 597
- Issue:
- 15
- ISSN:
- 0022-3751
- Page Range / eLocation ID:
- p. 3817-3832
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Arabidopsis thaliana Ca2+channel subunit CYCLIC NUCLEOTIDE‐GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+]cytsignalling in roots.cngc2 loss‐of‐function mutants were used:cngc2‐3 anddefence not death1 (which expresses cytosolic aequorin).cngc2 roots were significantly impaired. -
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Key points Vascular oxidative stress increases with advancing age.
We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H2O2.
H2O2‐induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from old
vs . young mice; the rise in vessel wall [Ca2+]iinduced by H2O2was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL‐10 mimicked the effects of advanced age on cell survival.Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis.
Vascular cells develop resilience to H2O2during ageing by preventing Ca2+overload and endothelial integrity promotes SMC survival.
Abstract Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H2O2. Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H2O2(200 µ
m , 50 min). Mouse superior epigastric arteries (SEAs; diameter, ∼150 µm) were isolated and pressurized to 100 cmH2O at 37˚C. For SEAs from young (4 months) mice, H2O2killed 57% of SMCs and 11% of ECs in malesvs . 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) mice, SMC death was reduced to 10% with diminished accumulation of [Ca2+]iin the vessel wall during H2O2exposure. In young mice, genetic deletion of IL‐10 mimicked the protective effect of ageing on cell death and [Ca2+]iaccumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µm ) increased SMC death, inhibiting NO synthase (l ‐NAME, 100 µm ) or scavenging peroxynitrite (FeTPPS, 5 µm ) reduced SMC death along with [Ca2+]i. Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z‐VAD‐FMK, 50 µm ) attenuated cell death with immunostaining for annexin V, cytochrome C, and caspases 3 and 9 pointing to induction of intrinsic apoptosis during H2O2exposure. We conclude that advanced age reduces Ca2+influx that triggers apoptosis, thereby promoting resilience of the vascular wall during oxidative stress. -
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Key points Accumulation of inorganic phosphate (P
i ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established.We showed that P
i promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR .Pre‐treatment of permeabilized myofibres with 2 m
m Cl− channel blocker 9‐anthracenecarboxylic acid (9AC) inhibited both effects of Pi .The biphasic dependence of Ca
2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra‐SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl− pathway of an as‐yet‐undefined molecular nature.Abstract Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P
i ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f /f ref ) rose with increasing [Pi ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi ] = 80 mm . Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9‐anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400 µm) applied to the cytoplasm produced a non‐significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55 mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue. -
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Key points Cardiac electrophysiology and Ca2+handling change rapidly during the fight‐or‐flight response to meet physiological demands.
Despite dramatic differences in cardiac electrophysiology, the cardiac fight‐or‐flight response is highly conserved across species.
In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+transients in innervated mouse and rabbit hearts.
Despite similar heart rate and Ca2+handling responses between mouse and rabbit hearts, we found notable species differences in spatio‐temporal repolarization dynamics during SNS.
Species‐specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine‐tuned across species to produce optimal cardiac fight‐or‐flight responses.
Abstract Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff‐perfused rabbit and mouse hearts. Dual optical mapping with voltage‐ and Ca2+‐sensitive dyes allowed for analysis of spatio‐temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at
t = 0 svs . 60.6 ± 4.1 ms att = 15 s,P < 0.05) followed by shortening (46.5 ± 9.1 ms att = 60 s,P = NSvs. t = 0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by β‐adrenergic activation. Simulations with species‐specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight‐or‐flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly. -
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Summary Low concentrations of CO2cause stomatal opening, whereas [CO2] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+and protein phosphorylation in CO2‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+into specific phosphorylation events. However, Ca2+‐binding proteins that function in the stomatal CO2response remain unknown.
Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed.
We isolated
cpk quintuple mutants and analyzed stomatal movements in response to CO2, light and abscisic acid (ABA). Interestingly, we found thatcpk3/5/6/11/23 quintuple mutant plants, but not other analyzedcpk quadruple/quintuple mutants, were defective in high CO2‐induced stomatal closure and, unexpectedly, also in low CO2‐induced stomatal opening. Furthermore, K+‐uptake‐channel activities were reduced incpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targetedcbl1/4/5/8/9 quintuple mutant plants.Our findings describe combinatorial
cpk mutants that function in CO2control of stomatal movements and support the results of classical studies showing a role for Ca2+in this response.
