Ion channel activities of cultured rat mesangial cells

1991 ◽  
Vol 261 (5) ◽  
pp. F808-F814 ◽  
Author(s):  
H. Matsunaga ◽  
N. Yamashita ◽  
Y. Miyajima ◽  
T. Okuda ◽  
H. Chang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Ion Channels ◽  
Patch Clamp ◽  
Ion Channel ◽  
Arginine Vasopressin ◽  
Mesangial Cells ◽  
Cation Channel ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Inside Out

We used the patch-clamp technique to clarify the nature of ion channels in renal mesangial cells in culture. In the cell-attached mode most patches were silent in the absence of agonists. In some patches a 25-pS nonselective channel was observed. This 25-pS cation channel was consistently observed in inside-out patches, and it was activated by intracellular Ca2+. Excised patch experiments also revealed the existence of a 40-pS K+ channel, which was activated by intracellular Ca2+. This 40-pS K+ channel was observed infrequently in the cell-attached mode. The activities of both channels were increased by arginine vasopressin or angiotensin II, resulting from an increase in intracellular Ca2+ concentration.

WHAT CAUSES ION CHANNEL PROTEINS TO FLUCTUATE OPEN AND CLOSED?

1996 ◽  
Vol 07 (04) ◽  
pp. 321-331 ◽  
Author(s):  
LARRY S. LIEBOVITCH ◽  
ANGELO T. TODOROV
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Ion Channel ◽  
Patch Clamp Technique ◽  
Sodium Potassium ◽  
Random Event ◽  
Channel Protein ◽  
Channel Proteins ◽  
Ion Channel Proteins ◽  
Open States

Ion channels in the cell membrane spontaneously switch from states that are closed to the flow of ions such as sodium, potassium, and chloride to states that are open to the flow of these ions. The durations of times that an individual ion channel protein spends in the closed and open states can be measured by the patch clamp technique. We explore two basic issues about the molecular properties of ion channels: 1) If the switching between the closed and open state is an inherently random event, what does the patch clamp data tell us about the structure or motions in the ion channel protein? 2) Is this switching random?

Patch-Clamp Recording of Ion Channels: Interfering Effects of Patch Pipette Glass

Physiology ◽  
1990 ◽  
Vol 5 (4) ◽  
pp. 155-158
Author(s):  
C Zuazaga ◽  
A Steinacker
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Ion Channel ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Ion Channel Properties ◽  
Patch Pipette ◽  
Channel Properties

Glass for pipettes used to record ion channel activity with the patch-clamp technique is selected on the basis of its electrical, thermal, and sealing properties. Recent findings stress a new characteristic to consider: the effect of pipette glass itself on ion channel properties.

scholarly journals A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid.

1992 ◽  
Vol 100 (6) ◽  
pp. 1021-1040 ◽  
Author(s):  
D Kim
Keyword(s):  
Arachidonic Acid ◽  
Ion Channels ◽  
Ion Channel ◽  
Mechanosensitive Channel ◽  
K Channel ◽  
Heart Cells ◽  
Dependent Manner ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Fatty Acid Binding

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.

scholarly journals Using the Patch-Clamp technique to shed light on ion channels structure, function and pharmacology

2014 ◽  
Author(s):  
Roberta Gualdani
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Transient Receptor Potential ◽  
Neuronal Excitability ◽  
Receptor Potential ◽  
Cellular Membrane ◽  
Bk Channel ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Transient Receptor

Ion channels are membrane proteins that selectively allow ions to flow down their electrochemical gradient across the cellular membrane. They localize in both plasma and intracellular membranes and regulate a variety of functions such as neuronal excitability, heartbeat, muscle contraction and hormones release. Thus, understanding the molecular mechanism of ion channels function and regulation is one of the key goals of modern Biophysics. During my PhD thesis, by combining patch-clamp measurements with site-direct mutagenesis, fluorophore labeling experiments and pharmacological assays, I explored some functional and structural properties of different ion transporters: the Na+/Ca2+ exchanger (NCX); the large conductance Ca2+-voltage activated K+ channel (BK) channel; the human Transient receptor potential, member A1 (TRPA1) channel.

Involvement of actin cytoskeleton in modulation of apical K channel activity in rat collecting duct

1994 ◽  
Vol 267 (4) ◽  
pp. F592-F598 ◽  
Author(s):  
W. H. Wang ◽  
A. Cassola ◽  
G. Giebisch
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Inhibition ◽  
Inside Out

We have employed the patch-clamp technique to investigate the role of the actin cytoskeleton in the modulation of the low-conductance K+ channel in the apical membrane of the rat cortical collecting duct (CCD). This K+ channel is inactivated by application of cytochalasin B or D, both compounds known to disrupt actin filaments. The effect of both cytochalasins, B and D, was fully reversible in cell-attached patches, but channel activity could not be fully restored in excised membrane patches. The effect of cytochalasins on channel activity was specific and resulted from depolymerization of the actin cytoskeleton, since application of 10 microM chaetoglobosin C, a cytochalasin analogue that does not depolymerize the actin filaments, had no effect on channel activity in inside-out patches. Addition of either actin monomers or of the polymerizing actin filaments in inside-out patches to the cytosolic medium had no effect on channel activity. This suggests that cytochalasin B- or D-induced inactivation of apical K+ channels is not caused by obstruction of the channel pore by actin. We also observed that channel inhibition by cytochalasin B or D could be blocked by pretreatment with 5 microM phalloidin, a compound that stabilizes actin filaments. We conclude that apical K+ channel activity depends critically on the integrity of the actin cytoskeleton.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

scholarly journals Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes

2015 ◽  
Vol 36 (3) ◽  
pp. 1049-1058 ◽  
Author(s):  
Lena Rubi ◽  
Vaibhavkumar S. Gawali ◽  
Helmut Kubista ◽  
Hannes Todt ◽  
Karlheinz Hilber ◽  
...  
Keyword(s):  
Ion Channels ◽  
Muscular Dystrophy ◽  
Ion Channel ◽  
Cardiac Electrophysiology ◽  
Cardiac Complications ◽  
Membrane Repair ◽  
Patch Clamp Technique ◽  
Cardiac Ion Channels ◽  
Ventricular Cardiomyocytes ◽  
Voltage Dependent

Background/Aims: Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B). Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. Methods and Results: By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf) mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Conclusion: Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types.

Two types of K+ channel in thick ascending limb of rat kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F599-F605 ◽  
Author(s):  
W. H. Wang
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inside Out

We have used the patch-clamp technique to study the apical K+ channels in the thick ascending limb (TAL) of the rat kidney. Two types of K+ channels, a low-conductance and an intermediate-conductance K+ channel, were identified in both cell-attached and inside-out patches. We confirmed the previously reported intermediate-conductance K+ channel (72 pS), which is inhibited by millimolar cell ATP, acidic pH, Ba2+, and quinidine (4). We now report a second K+ channel in apical membrane of the TAL. The slope conductance of this low-conductance K+ channel is 30 pS, and its open probability is 0.80 in cell-attached patches. This channel is not voltage dependent, and application of 2 mM ATP in the bath inhibits channel activity in inside-out patches. In addition, 250 microM glyburide, an ATP-sensitive K+ channel inhibitor, blocks channel activity, whereas the same concentration of glyburide has no inhibitory effect on the 72-pS K+ channel. Channel activity of the 30-pS K+ channel decreases rapidly upon excision of patches (channel run down). Application of 0.1 mM ATP and the catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) restores channel activity. Furthermore, addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP or 50-100 pM vasopressin in the cell-attached patches increases channel activity. In conclusion, two types of K+ channels are present in the apical membrane of TAL of rat kidney, and PKA plays an important role in modulation of the low-conductance K+ channel activity.

Calcium-modulated inward rectification of a calcium-activated potassium channel in neurons

1994 ◽  
Vol 72 (6) ◽  
pp. 3023-3025 ◽  
Author(s):  
J. Kang ◽  
C. Sumners ◽  
P. Posner
Keyword(s):  
Patch Clamp ◽  
Brain Stem ◽  
Inward Rectification ◽  
K Channel ◽  
Minor Effect ◽  
Bath Solution ◽  
Old Rats ◽  
A Minor ◽  
Calcium Activated Potassium Channel ◽  
Inside Out

1. Inward rectification of a calcium-activated K+ channel in neurons cultured from the hypothalamus and brain stem of 1-day-old rats was studied by using patch-clamp techniques. A big conductance calcium-activated K+ channel with a slow gating rate was observed in inside-out patches. With symmetrical K+ across patches, inward conductance of this calcium-activated K+ channel was 216 +/- 14 (SE) pS (n = 4 patches), which changed little as different [Ca2+] was included in the bath solution. Outward conductance of this calcium-activated K+ channel was regulated by [Ca2+] in the bath solution and was 74 +/- 15 pS with 500 microM Ca2+. The higher level of Ca2+ on the intracellular side of the membrane caused the larger degree of rectification. Mg2+ only had a minor effect on rectification of this calcium-activated K+ channel.

scholarly journals Development of a Novel Automated Ion Channel Recording Method Using “Inside-Out” Whole-Cell Membranes

2005 ◽  
Vol 10 (8) ◽  
pp. 806-813 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Thomas L. Merrill ◽  
Mark R. Bowlby
Keyword(s):  
Ion Channels ◽  
Parallel Implementation ◽  
Drug Application ◽  
K Channel ◽  
Positive Pressure ◽  
Screening Methods ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Configuration ◽  
Inside Out

Efforts to develop novelmethods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique “inside-out” whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cellmay be moved through the air, first rupturing part of the cellularmembrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, aswell as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound inactivities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.

Sign in / Sign up

Export Citation Format

Share Document