L-type Ca2+ channel function in the avian embryonic heart after cardiac neural crest ablation

2005 ◽  
Vol 288 (3) ◽  
pp. H1173-H1178 ◽  
Author(s):  
Carol A. Nichols ◽  
Tony L. Creazzo
Keyword(s):  
Neural Crest ◽  
Single Channel ◽  
Persistent Truncus Arteriosus ◽  
Surgical Ablation ◽  
Time Frequency ◽  
Cardiac Neural Crest ◽  
Channel Function ◽  
Open Time ◽  
Mammalian Embryos ◽  
The Mean

In avian and mammalian embryos, surgical ablation or severely reduced migration of the cardiac neural crest leads to a failure of outflow tract septation known as persistent truncus arteriosus (PTA) and leads to embryo lethality due partly to impaired excitation-contraction coupling stemming primarily from a reduction in the L-type Ca2+ current ( ICa,L). Decreased ICa,L occurs without a corresponding reduction in the α1-subunit of the Ca2+ channel. We hypothesize that decreased ICa,L is due to reduced function at the single channel level. The cell-attached patch clamp with Na+ as the charge carrier was used to examine single Ca2+ channel activity in myocytes from normal hearts from sham-operated embryos and from hearts diagnosed with PTA at embryonic days (ED) 11 and 15 after laser ablation of the cardiac neural crest. In normal hearts, the number of single channel events per 200-ms depolarization and the mean open channel probability ( Po) was 1.89 ± 0.17 and 0.067 ± 0.008 for ED11 and 1.14 ± 0.17 and 0.044 ± 0.005 for ED15, respectively. These values represent a normal reduction in channel function and ICa,L observed with development. However, the number of single channel events was significantly reduced in hearts with PTA at both ED11 and ED15 (71% and 47%, respectively) with a corresponding reduction in Po (75% and 43%). The open time frequency histograms were best fitted by single exponentials with similar decay constants (τ ≅ 4.5 ms) except for the sham operated at ED15 (τ = 3.4 ms). These results indicate that the cardiac neural crest influences the development of myocardial Ca2+ channels.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

Basolateral potassium channels in renal proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F476-F487 ◽  
Author(s):  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
K Channels ◽  
Open Time ◽  
Excised Patches ◽  
The Mean ◽  
Inside Out

Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).

scholarly journals Cardiac neural crest ablation results in early endocardial cushion and hemodynamic flow abnormalities

2016 ◽  
Vol 311 (5) ◽  
pp. H1150-H1159 ◽  
Author(s):  
Pei Ma ◽  
Shi Gu ◽  
Ganga H. Karunamuni ◽  
Michael W. Jenkins ◽  
Michiko Watanabe ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Neural Crest ◽  
Late Stage ◽  
Heart Defects ◽  
Neural Crest Cell ◽  
Vessel Diameter ◽  
Double Outlet Right Ventricle ◽  
Great Vessel ◽  
Persistent Truncus Arteriosus ◽  
Cardiac Neural Crest

Cardiac neural crest cell (CNCC) ablation creates congenital heart defects (CHDs) that resemble those observed in many syndromes with craniofacial and cardiac consequences. The loss of CNCCs causes a variety of great vessel defects, including persistent truncus arteriosus and double-outlet right ventricle. However, because of the lack of quantitative volumetric measurements, less severe defects, such as great vessel size changes and valve defects, have not been assessed. Also poorly understood is the role of abnormal cardiac function in the progression of CNCC-related CHDs. CNCC ablation was previously reported to cause abnormal cardiac function in early cardiogenesis, before the CNCCs arrive in the outflow region of the heart. However, the affected functional parameters and how they correlate with the structural abnormalities were not fully characterized. In this study, using a CNCC-ablated quail model, we contribute quantitative phenotyping of CNCC ablation-related CHDs and investigate abnormal early cardiac function, which potentially contributes to late-stage CHDs. Optical coherence tomography was used to assay early- and late-stage embryos and hearts. In CNCC-ablated embryos at four-chambered heart stages, great vessel diameter and left atrioventricular valve leaflet volumes are reduced. Earlier, at cardiac looping stages, CNCC-ablated embryos exhibit abnormally twisted bodies, abnormal blood flow waveforms, increased retrograde flow percentage, and abnormal cardiac cushions. The phenotypes observed in this CNCC-ablation model were also strikingly similar to those found in an established avian fetal alcohol syndrome model, supporting the contribution of CNCC dysfunction to the development of alcohol-induced CHDs.

scholarly journals Gating of Acid-sensitive Ion Channel-1: Release of Ca2+ Block vs. Allosteric Mechanism

2006 ◽  
Vol 127 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Ping Zhang ◽  
Fred J. Sigworth ◽  
Cecilia M. Canessa
Keyword(s):  
Open Channel ◽  
Channel Blocker ◽  
Single Channel ◽  
Allosteric Mechanism ◽  
Open Time ◽  
Channel Opening ◽  
Voltage Dependent ◽  
High Affinity Binding Site ◽  
The Mean ◽  
High Concentrations

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at −60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.

Calcium currents in hearts with persistent truncus arteriosus

1992 ◽  
Vol 262 (4) ◽  
pp. H1182-H1190 ◽  
Author(s):  
S. Aiba ◽  
T. L. Creazzo
Keyword(s):  
Neural Crest ◽  
Heart Development ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Large Fraction ◽  
Calcium Currents ◽  
Cholinergic Innervation ◽  
Truncus Arteriosus ◽  
Embryonic Chick ◽  
Persistent Truncus Arteriosus

We have used the neural crest model of defective heart development to characterize both L- and T-type Ca2+ currents (ICa,L and ICa,T) in ventricular myocytes from embryonic chick hearts with a severe outflow tract anomaly known as persistent truncus arteriosus (PTA). Because of smaller whole embryo weights but no significant change in the weights of ventricles with PTA, the ventricle to whole embryo weight ratios from hearts with PTA were 61% larger than normal at day 11 of incubation. There was a 51% reduction in the peak magnitude of ICa,L at a test potential of +10 mV (-1.4 vs. -0.7 microA/cm2), whereas ICa,T, a proportionately large fraction of the total Ca2+ current in the embryonic chick ventricle, was unaffected. In comparison to sham-operated controls, ICa,L was otherwise not different. Half-activation occurred at about -1 and -41 mV, whereas half-inactivation occurred at -19 and -61 mV for ICa,L and ICa,T, respectively. The time for half-recovery from inactivation were not different and were 200 and 230 ms for ICa,L and ICa,T, respectively. The time for half-decay of the currents and their responses to BAY K 8644 were also similar in both sham-operated and experimental hearts. Although the dihydropyridine receptor binding experiments suggest that the total number of L-type Ca2+ channels was not different, the results from the physiological experiments indicate that the number of functional L-type channels available for opening and/or the single-channel conductance may be reduced in hearts with PTA. Finally, our results with the neural crest model indicate that it is unlikely that the development of Ca2+ currents is influenced by the onset of cholinergic innervation in the heart.

Fate of the mammalian cardiac neural crest

Development ◽  
2000 ◽  
Vol 127 (8) ◽  
pp. 1607-1616 ◽  
Author(s):  
X. Jiang ◽  
D.H. Rowitch ◽  
P. Soriano ◽  
A.P. McMahon ◽  
H.M. Sucov
Keyword(s):  
Neural Crest ◽  
Genetic System ◽  
Outflow Tract ◽  
Substantial Contribution ◽  
Pharyngeal Arch ◽  
Postnatal Life ◽  
Cardiac Neural Crest ◽  
Mammalian Embryos ◽  
Pharyngeal Arch Arteries

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: Chloride imaging and cell-attached patch-clamp recordings

2000 ◽  
Vol 17 (2) ◽  
pp. 197-206 ◽  
Author(s):  
WALLACE B. THORESON ◽  
RON NITZAN ◽  
ROBERT F. MILLER
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Open Time ◽  
The Mean ◽  
Channel Properties ◽  
Distribution Fit

The present study uses cell-attached patch-recording techniques to study the single-channel properties of Ca2+ channels in isolated salamander photoreceptors and investigate their sensitivity to reductions in intracellular Cl−. The results show that photoreceptor Ca2+ channels possess properties similar to L-type Ca2+ channels in other preparations, including (1) enhancement of openings by the dihydropyridine agonist, (−)BayK8644; (2) suppression by a dihydropyridine antagonist, nisoldipine; (3) single-channel conductance of 22 pS with 82 mM Ba2+ as the charge carrier; (4) mean open probability of 0.1; (5) open-time distribution fit with a single exponential (τ0 = 1.1 ms) consistent with a single open state; and (6) closed time distribution fit with two exponentials (τc1 = 0.7 ms, τc2 = 25.4 ms) consistent with at least two closed states. Using a Cl−-sensitive dye to measure intracellular [Cl−], it was found that perfusion with gluconate-containing, low Cl− medium depleted intracellular [Cl−]. It was therefore possible to reduce intracellular [Cl−] by perfusion with a low Cl− solution while maintaining the extracellular channel surface in high Cl− pipette solution. Under these conditions, the single-channel conductance was unchanged, but the mean open probability fell to 0.03. This reduction can account for the 66% reduction in whole-cell Ca2+ currents produced by perfusion with low Cl− solutions. Examination of the open and closed time distributions suggests that the reduction in open probability arises from increases in closed-state dwell times. Changes in intracellular [Cl−] may thus modulate photoreceptor Ca2+ channels.

Characterization of an amiloride binding region in the α-subunit of ENaC

2003 ◽  
Vol 285 (6) ◽  
pp. F1279-F1290 ◽  
Author(s):  
Ollie Kelly ◽  
Chaomei Lin ◽  
Mohan Ramkumar ◽  
Nina C. Saxena ◽  
Thomas R. Kleyman ◽  
...  
Keyword(s):  
Single Channel ◽  
Point Mutations ◽  
Binding Affinities ◽  
Extracellular Loop ◽  
Wild Type ◽  
Α Subunit ◽  
Open Time ◽  
The Mean ◽  
Α Subunits

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the α-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278–283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type α-subunits or α-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities ( Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high- Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type α-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.

scholarly journals How ATP Inhibits the Open KATP Channel

2008 ◽  
Vol 132 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Tim J. Craig ◽  
Frances M. Ashcroft ◽  
Peter Proks
Keyword(s):  
Binding Site ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Open Time ◽  
Mechanism Of Interaction ◽  
The Mean ◽  
Kinetics Of

ATP-sensitive potassium (KATP) channels are composed of four pore-forming Kir6.2 subunits and four regulatory SUR1 subunits. Binding of ATP to Kir6.2 leads to inhibition of channel activity. Because there are four subunits and thus four ATP-binding sites, four binding events are possible. ATP binds to both the open and closed states of the channel and produces a decrease in the mean open time, a reduction in the mean burst duration, and an increase in the frequency and duration of the interburst closed states. Here, we investigate the mechanism of interaction of ATP with the open state of the channel by analyzing the single-channel kinetics of concatenated Kir6.2 tetramers containing from zero to four mutated Kir6.2 subunits that possess an impaired ATP-binding site. We show that the ATP-dependent decrease in the mean burst duration is well described by a Monod-Wyman-Changeux model in which channel closing is produced by all four subunits acting in a single concerted step. The data are inconsistent with a Hodgkin-Huxley model (four independent steps) or a dimer model (two independent dimers). When the channel is open, ATP binds to a single ATP-binding site with a dissociation constant of 300 μM.

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