Stretch-activated ion channels in tissue-cultured chick heart

1993 ◽  
Vol 264 (3) ◽  
pp. H960-H972 ◽  
Author(s):  
A. Ruknudin ◽  
F. Sachs ◽  
J. O. Bustamante
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Inward Rectifier ◽  
Channel Noise ◽  
Patch Clamp Recording ◽  
Alkali Cations ◽  
Channel Expression ◽  
Inward Currents ◽  
Extracellular Ions ◽  
Stretch Activated Ion Channels

With use of single-channel patch-clamp recording, we found five distinct types of stretch-activated ion channels (SACs) in tissue-cultured embryonic chick cardiac myocytes. With 140 mM K+ saline in the pipette, four channels had linear conductances of approximately equal to 25, 50, 100, and 200 pS and other channel was an inward rectifier of approximately equal to 25 pS at 0 mV membrane potential. The 100- and 200-pS channels were K+ selective, whereas the others passed alkali cations and Ca2+. From reversal potentials, the permeability ratio of K+/Na+, PK/PNa, was 3–7 for nonselective channels and 7–16 for K(+)-selective channels. Channel density was approximately equal to 0.3/microns2 for linear conductances and approximately equal to 0.1/microns2 for inward rectifier. Open-channel noise was a function of pipette filling solution with root-mean-square (RMS) noise increasing in the order K+ < isosmotic sucrose (plus trace ions) < Na+, probably reflecting short-lived block by extracellular ions. All channels were blocked by 20 microM Gd3+. The 25-pS linear channel was also blocked by 12.5 microM tetrodotoxin and 10 microM diltiazem, but the others were insensitive at these concentrations. Extracellular Cs+ and tetraethylammonium chloride did not block any channels. We saw no SAC activity in cells grown without embryo extract (EE), which demonstrates that channel expression, or some necessary cofactor, is under control of growth factors. Basic fibroblast growth factor (FGF) could replace EE in supporting channel expression. The presence of SACs capable of generating inward currents might explain how stretch increases automaticity in the heart. Because some SACs were permeable to Ca2+, they could contribute to the Starling curve and perhaps to initiating stretch-induced hypertrophy.

Voltage-gated currents of rabbit A- and B-type horizontal cells in retinal monolayer cultures

1994 ◽  
Vol 11 (2) ◽  
pp. 369-378 ◽  
Author(s):  
Stefan Löhrke ◽  
Hans-Dieter Hofmann
Keyword(s):  
Single Channel ◽  
Ionic Currents ◽  
Calcium Currents ◽  
Horizontal Cells ◽  
Delayed Rectifier ◽  
Patch Clamp Recording ◽  
Voltage Gated ◽  
Monolayer Cultures ◽  
Inward Currents

AbstractIn monolayer cultures prepared from immature early postnatal rabbit retina, small populations of neurons can be demonstrated to differentiate into apparently mature A- and B-type horizontal cells. Using wholecell, single-channel, patch-clamp recording techniques, we have analyzed the pattern of voltage-gated conductances expressed by mammalian horizontal cells under these conditions. A total of six different voltage-dependent ionic currents were recorded. Tetrodotoxin-sensitive fast sodium inward currents (INa) were found in 81% of the A-type and 90% of the B-type cells. Inward calcium currents could be demonstrated in all cells tested after blockade of other conductances. Two types of outward potassium currents with properties of the 4–aminopyridine-sensitive transient IA and the tetraethylammonium sensitive delayed rectifier IK, respectively, could be characterized in whole-cell recordings. An inward rectifying potassium current (Ianom) typical for horizontal cells was activated in response to hyperpolarizing voltage steps. These types of currents have also been described in dissociated adult horizontal cells from lower vertebrates and cat. With single-channel recordings on inside-out patches excised from B-type cells, an additional Ca2+-dependent current (IK(Ca)) was observed which, so far, has not been described in horizontal cells developing in situ. Our results demonstrate that cultured rabbit horizontal cells express a set of voltage-gated currents which largely, but not completely, corresponds to that described in situ for horizontal cells of other species. The culture system will allow further investigation of developmental and functional aspects of mammalian horizontal cells.

scholarly journals Functional Roles of Charged Amino Acid Residues on the Wall of the Cytoplasmic Pore of Kir2.1

2006 ◽  
Vol 127 (4) ◽  
pp. 401-419 ◽  
Author(s):  
Yuichiro Fujiwara ◽  
Yoshihiro Kubo
Keyword(s):  
Amino Acids ◽  
Negative Charge ◽  
Single Channel ◽  
Outward Current ◽  
Channel Noise ◽  
Inward Rectification ◽  
K Channel ◽  
Patch Clamp Recording ◽  
Charged Amino Acids ◽  
Charged Residues

It is known that rectification of currents through the inward rectifier K+ channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg2+ and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K+ permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg2+ or spermine and the voltage dependence of the blockade. It also reduced K+ permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg2+, spermine, and K+ so that the transmembrane pore is sufficiently filled with K+ ions, which enables strong voltage-dependent blockade with adequate outward K+ conductance.

Acid-sensing ion channels contribute to the increase in vesicular release from SH-SY5Y cells stimulated by extracellular protons

2012 ◽  
Vol 303 (4) ◽  
pp. C376-C384 ◽  
Author(s):  
Qiu-Ju Xiong ◽  
Zhuang-Li Hu ◽  
Peng-Fei Wu ◽  
Lan Ni ◽  
Zhi-Fang Deng ◽  
...  
Keyword(s):  
Ion Channels ◽  
Neurotransmitter Release ◽  
Neuronal Cell ◽  
Channel Blockers ◽  
Patch Clamp Recording ◽  
Human Neuroblastoma ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
Vesicular Release ◽  
Inward Currents

Acid-sensing ion channels (ASICs) have been reported to play a role in the neuronal dopamine pathway, but the exact role in neurotransmitter release remains elusive. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line, which can release monoamine neurotransmitters. In this study, the expression of ASICs was identified in SH-SY5Y cells to further explore the role of ASICs in vesicular release stimulated by acid. We gathered evidence that ASICs could be detected in SH-SY5Y cells. In whole cell patch-clamp recording, a rapid decrease in extracellular pH evoked inward currents, which were reversibly inhibited by 100 μM amiloride. The currents were pH dependent, with a pH of half-maximal activation (pH0.5) of 6.01 ± 0.04. Furthermore, in calcium imaging and FM 1-43 dye labeling, it was shown that extracellular protons increased intracellular calcium levels and vesicular release in SH-SY5Y cells, which was attenuated by PcTx1 and amiloride. Interestingly, N-type calcium channel blockers inhibited the vesicular release induced by acidification. In conclusion, ASICs are functionally expressed in SH-SY5Y cells and involved in vesicular release stimulated by acidification. N-type calcium channels may be involved in the increase in vesicular release induced by acid. Our results provide a preliminary study on ASICs in SH-SY5Y cells and neurotransmitter release, which helps to further investigate the relationship between ASICs and dopaminergic neurons.

Inward rectifier K+ channel from human heart and brain: cloning and stable expression in a human cell line

1995 ◽  
Vol 268 (1) ◽  
pp. H506-H511
Author(s):  
M. D. Ashen ◽  
B. O'Rourke ◽  
K. A. Kluge ◽  
D. C. Johns ◽  
G. F. Tomaselli
Keyword(s):  
Cell Line ◽  
Human Cell ◽  
Single Channel ◽  
Human Cell Line ◽  
Inward Rectifier ◽  
K Channel ◽  
Steady State Current ◽  
Channel Expression ◽  
Novel Strategy ◽  
Human And Mouse

We have cloned the human homologue of the inward rectifier K+ channel from both heart and brain tissue (HHBIRK1). The human clones were identical to each other in their coding regions and were highly homologous to the mouse macrophage (IRK1) channel. The inward rectifier currents from human and mouse clones were characterized using a novel strategy for stable ion channel expression in a human cell line. The permeability of the expressed inwardly rectifying channels was greater for K+ than for Rb+, whereas no current was observed when K+ was replaced by Na+. A prominent time- and voltage-dependent block was observed in the presence of Ba2+, whereas a small decay in the steady-state current was observed with millimolar concentrations of Na+. Single-channel conductances of 49.1 +/- 3.3 pS (n = 6) and 40.2 +/- 2.5 pS (n = 3) (P = 0.005) were obtained for the HHBIRK1 and IRK1 clones, respectively. These results indicate that sequence dissimilarities between human and mouse inward rectifier K+ channels may have significant functional consequences.

scholarly journals Characteristics of Five Types of K+ Channel in Cultured Locust Muscle

1990 ◽  
Vol 154 (1) ◽  
pp. 45-65
Author(s):  
P. N. Usherwood
Keyword(s):  
Single Channel ◽  
Inward Rectifier ◽  
Resting Potential ◽  
K Channel ◽  
Chord Conductance ◽  
Outward Currents ◽  
Inward Currents

K+ channel activity in cultured locust myofibres was investigated using gigaohm patch-clamp techniques. After 2 months in vitro the myofibres had a mean resting potential of −39 ± 7 mV (±S.D., 7V = 42). Five types of K+ channel were identified at this time. The majority of single-channel events recorded from cellattached patches were due to a small-conductance (type 1) and a largeconductance (type 2), inward rectifier, K+ channel. In cell-attached patches, with 180 mmoll−1 KC1 in the patch pipette, the type 1 channel had a chord conductance of 43 pS for inward currents and 8pS for outward currents; the type 2 channel had a chord conductance of 115 pS for inward currents and 29 pS for outward currents. The type 2 channel exhibited bursting kinetics, was ATP-sensitive and could be blocked by Ba2+. Two other channels (types 3 and 4) had linear conductances of 130pS and 207pS, respectively. The type 3 channel was Ca2+-sensitive. A further channel (type 5) appeared to be an inward rectifier with a conductance of 5pS. Openings of types 3, 4 and 5 channels occurred less frequently than openings of the other two channels. Types 1, 2, 3 and 4 channels possessed multiple closed and open states with non-linear gating mechanisms.

scholarly journals CellSpecks: A Software for Automated Detection and Analysis of Calcium Channels in Live Cells

2018 ◽  
Author(s):  
S I Shah ◽  
M Smith ◽  
D Swaminathan ◽  
I Parker ◽  
G Ullah ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Nicotinic Acetylcholine Receptors ◽  
High Throughput Screening ◽  
Single Channel ◽  
Video Recording ◽  
Synthetic Data ◽  
Live Cells ◽  
Patch Clamp Recording ◽  
Additional Advantage

ABSTRACTTo couple the fidelity of patch-clamp recording with a more high-throughput screening capability, we pioneered a novel approach to single channel recording that we named “optical patch clamp”. By using highly-sensitive fluorescent Ca2+ indicator dyes in conjunction with total internal fluorescence microscopy techniques, we monitor Ca2+ flux through individual Ca2+-permeable channels. This approach provides information about channel gating analogous to patch-clamp recording at time resolution of ~ 2 ms, with the additional advantage of being massively parallel, providing simultaneous and independent recording from thousands of channels in native environment. However, manual analysis of the data generated by this technique presents severe challenges as a video recording can include many thousands of frames. To overcome this bottleneck, we developed an image processing and analysis framework called CellSpecks, capable of detecting and fully analyzing the kinetics of ion channels within a video sequence. By using a randomly generated synthetic data, we tested the ability of CellSpecks to rapidly and efficiently detect and analyze the activity of thousands of ion channels, including openings for a few milliseconds. Here, we report the use of CellSpecks for the analysis of experimental data acquired by imaging muscle nicotinic acetylcholine receptors and the Alzheimer’s disease-associated amyloid beta pores with multiconductance levels in the plasma membrane of Xenopus laevis oocytes. We show that CellSpecks can accurately and efficiently generate location maps, create raw and processed fluorescence time-traces, histograms of mean open times, mean close times, open probabilities, durations, and maximum amplitudes, and a ‘channel chip’ showing the activity of all channels as a function of time. Although we specifically illustrate the application of CellSpecks for analyzing data from Ca2+ channels, it can be easily customized to analyze other spatially and temporally localized signals.

Differential expression of ORCC and CFTR induced by low temperature in CF airway epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C243-C251 ◽  
Author(s):  
M. E. Egan ◽  
E. M. Schwiebert ◽  
W. B. Guggino
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Incubation Temperature ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Cl Channel ◽  
Channel Expression

When nonepithelial cell types expressing the delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) mutation are grown at reduced temperatures, the mutant protein can be properly processed. The effect of low temperatures on Cl- channel activity in airway epithelial cells that endogenously express the delta F508-CFTR mutation has not been investigated. Therefore, we examined the effect of incubation temperature on both CFTR and outwardly rectifying Cl- channel (ORCC) activity in normal, in cystic fibrosis (CF)-affected, and in wild-type CFTR-complemented CF airway epithelia with use of a combination of inside-out and whole cell patch-clamp recording, 36Cl- efflux assays, and immunocytochemistry. We report that incubation of CF-affected airway epithelial cells at 25-27 degrees C is associated with the appearance of a protein kinase A-stimulated CFTR-like Cl- conductance. In addition to the appearance of CFTR Cl- channel activity, there is, however, a decrease in the number of active ORCC when cells are grown at 25-27 degrees C, suggesting that the decrease in incubation temperature may be associated with multiple alterations in ion channel expression and/or regulation in airway epithelial cells.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

scholarly journals Single-channel Ca2+ imaging implicates Aβ1–42 amyloid pores in Alzheimer’s disease pathology

2011 ◽  
Vol 195 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Angelo Demuro ◽  
Martin Smith ◽  
Ian Parker
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Ion Channels ◽  
Single Channel ◽  
Membrane Integrity ◽  
Cytosolic Calcium ◽  
Xenopus Laevis Oocytes ◽  
Permeable Membrane ◽  
Membrane Pores ◽  
Pathological Mechanism

Oligomeric forms of Aβ peptides are implicated in Alzheimer’s disease (AD) and disrupt membrane integrity, leading to cytosolic calcium (Ca2+) elevation. Proposed mechanisms by which Aβ mediates its effects include lipid destabilization, activation of native membrane channels, and aggregation of Aβ into Ca2+-permeable pores. We distinguished between these using total internal reflection fluorescence (TIRF) microscopy to image Ca2+ influx in Xenopus laevis oocytes. Aβ1–42 oligomers evoked single-channel Ca2+ fluorescence transients (SCCaFTs), which resembled those from classical ion channels but which were not attributable to endogenous oocyte channels. SCCaFTs displayed widely variable open probabilities (Po) and stepwise transitions among multiple amplitude levels reminiscent of subconductance levels of ion channels. The proportion of high Po, large amplitude SCCaFTs grew with time, suggesting that continued oligomer aggregation results in the formation of highly toxic pores. We conclude that formation of intrinsic Ca2+-permeable membrane pores is a major pathological mechanism in AD and introduce TIRF imaging for massively parallel single-channel studies of the incorporation, assembly, and properties of amyloidogenic oligomers.

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