Basolateral membrane potassium channels in rabbit cortical thick ascending limb

1992 ◽  
Vol 263 (2) ◽  
pp. F262-F267 ◽  
Author(s):  
A. M. Hurst ◽  
M. Duplain ◽  
J. Y. Lapointe
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collagenase Treatment ◽  
Voltage Dependent ◽  
Selective Channel

The nature of K exit across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) was investigated using the patch clamp technique. The basolateral membrane was exposed by mild collagenase treatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductance was 35.0 +/- 1.3 pS (n = 9) compared with an outward conductance of 7.0 +/- 0.9 pS (n = 5), and the current reversed at a pipette potential of -63.5 +/- 3.1 mV (n = 9). The channel is strongly voltage dependent, showing an e-fold increase in open probability per 18-mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channel current amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excised patch recordings were possible, which confirmed the presence of a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possible to obtain patch clamp recordings from the rabbit CTAL basolateral membrane using a very mild collagenase treatment, and 2) the exit of K across the basolateral membrane is mediated at least in part by the presence of voltage-sensitive K channels.

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb

2002 ◽  
Vol 283 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
Rui-Min Gu ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Cytochrome P 450

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( P o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NP o, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

1999 ◽  
Vol 277 (5) ◽  
pp. C913-C925 ◽  
Author(s):  
Thierry van den Abbeele ◽  
Jacques Teulon ◽  
Patrice Tran Ba Huy
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Basolateral Membrane ◽  
Outer Hair Cells ◽  
Catalytic Subunit ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Guinea Pig Cochlea ◽  
Voltage Dependent ◽  
Inside Out

Cell-attached and cell-free configurations of the patch-clamp technique were used to investigate the conductive properties and regulation of the major K+channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major voltage-dependent K+ channels. A Ca2+-activated K+ channel with a high conductance (220 pS, P K/ P Na= 8) was found in almost 20% of the patches. The inside-out activity of the channel was increased by depolarizations above 0 mV and increasing the intracellular Ca2+concentration. External ATP or adenosine did not alter the cell-attached activity of the channel. The open probability of the excised channel remained stable for several minutes without rundown and was not altered by the catalytic subunit of protein kinase A (PKA) applied internally. The most frequent K+ channel had a low conductance and a small outward rectification in symmetrical K+ conditions (10 pS for inward currents and 20 pS for outward currents, P K/ P Na= 28). It was found significantly more frequently in cell-attached and inside-out patches when the pipette contained 100 μM acetylcholine. It was not sensitive to internal Ca2+, was inhibited by 4-aminopyridine, was activated by depolarization above −30 mV, and exhibited a rundown after excision. It also had a slow inactivation on ensemble-averaged sweeps in response to depolarizing pulses. The cell-attached activity of the channel was increased when adenosine was superfused outside the pipette. This effect also occurred with permeant analogs of cAMP and internally applied catalytic subunit of PKA. Both channels could control the cell membrane voltage of outer hair cells.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

Basolateral outward rectifier chloride channel in isolated crypts of mouse colon

2000 ◽  
Vol 279 (2) ◽  
pp. G277-G287 ◽  
Author(s):  
Olivier Mignen ◽  
Stéphane Egee ◽  
Martine Liberge ◽  
Brian J. Harvey
Keyword(s):  
Protein Kinase ◽  
Chloride Channels ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Open Probability ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Inside Out

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between −100 and −40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I− > SCN− > Br−> Cl− > NO3 − > F−≫ SO4 2− ≈ gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca2+ concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5′- O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5′- O-(2-thiodiphosphate) reactivated the channel.

A voltage-dependent and calcium-permeable ion channel in fused presynaptic terminals of Torpedo

1996 ◽  
Vol 75 (5) ◽  
pp. 1858-1870 ◽  
Author(s):  
A. Meir ◽  
R. Rahamimoff
Keyword(s):  
Ion Channel ◽  
Nerve Terminal ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Voltage Range ◽  
Voltage Dependent ◽  
The Mean ◽  
Presynaptic Nerve Terminals ◽  
Channel Open Time

1. We used a preparation of fused presynaptic nerve terminals of Torpedo electromotor nerve and the patch-clamp technique for characterization of single ion channels. We report here of a large, nonselective ion channel which is highly voltage dependent. 2. The slope conductance of the I-V relation was estimated by either direct measurement of the single-channel current amplitude at different voltages (850 +/- 18 pS (SE); n = 9) or by variance analysis (834 +/- 23 pS; n = 5). 3. The voltage dependence was examined in three ways. At steady-state DC voltage conditions, NPo (the open probability times the number of channels in the patch) was estimated. At potentials < 0 mV, the probability of the channel to open is negligible and increases dramatically, within a very narrow voltage range, to > 50% at +8 mV (n = 8). 4. In pulse experiments, the activation time delay is shorter as the voltage step reaches more positive values. The mean time for half activation (T1/2) decreases from 15 ms at +10 mV to 4 ms at +30 mV (n = 5). 5. Ensemble currents exhibit rectification in response to voltage ramps at negative potentials (n = 10). 6. The channel was found to be nonselective. Its permeability to Na+, K+, Cl-, glutamate, Ba+2, and Ca+2, relative to Na+, was 1.00, 1.00, 1.22, 1.07, 0.85, and 0.62, respectively. 7. Based on the transport number of calcium, the calculated driving force, and the mean channel open time, we estimated the number of calcium ions entering the nerve terminal upon depolarization. This number is not substantially different from the number of ions entering through voltage-dependent, calcium-selective channels in other cells. 8. We speculate that this nonselective ion channel, may serve as a calcium entry route into the nerve terminal and hence be involved in transmitter release.

scholarly journals Heterogeneity and Remodeling of Ion Currents in Cultured Right Atrial Fibroblasts From Patients With Sinus Rhythm or Atrial Fibrillation

2021 ◽  
Vol 12 ◽  
Author(s):  
Dorothee Jakob ◽  
Alexander Klesen ◽  
Elisa Darkow ◽  
Fabian A. Kari ◽  
Friedhelm Beyersdorf ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Patch Clamp ◽  
Sinus Rhythm ◽  
Cardiac Electrophysiology ◽  
Single Channel ◽  
Right Atrial ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cultured Fibroblasts ◽  
Outward Currents

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

scholarly journals Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

2002 ◽  
Vol 120 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Ion Channels ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Acid Sensing Ion Channels ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetics Of ◽  
Channel Properties

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small.

Change in K+ current of HeLa cells with progression of the cell cycle studied by patch-clamp technique

1993 ◽  
Vol 265 (2) ◽  
pp. C328-C336 ◽  
Author(s):  
A. Takahashi ◽  
H. Yamaguchi ◽  
H. Miyamoto
Keyword(s):  
Cell Cycle ◽  
Patch Clamp ◽  
Single Channel ◽  
Membrane Potentials ◽  
S Phase ◽  
Inward Rectification ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Fixed Membrane

The K+ channel of HeLa S3 cells in metaphase was analyzed by inside-out and whole cell patch-clamp techniques. The channel had the characteristics of strong inward rectification, small conductance (22 pS at -100 mV), and dependence on intracellular Ca2+. We investigated the cell cycle dependency of the channel, using cells synchronized by harvesting them at the mitotic stage. The cell capacitance increased gradually with increases in the cell volume toward the S phase. The inward K+ currents through the channel at fixed membrane potentials were highest in early G1 and then decreased with time to a minimum in the S phase, increasing again in the M phase. The permeabilities at fixed membrane potentials were also highest in early G1, decreased to minima in the S phase, and increased again toward the next mitosis. In contrast, mean amplitude and the open probability of the single channel at a fixed membrane potential (-60 mV) did not change significantly during the cell cycle. Therefore the capacitance increases with progression of the cell cycle, whereas the permeability decreases from early G1 to an apparent minimum in the S phase. These changes may be caused by cell cycle-dependent changes in the number of channels.

scholarly journals Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR

2002 ◽  
Vol 282 (1) ◽  
pp. L135-L145 ◽  
Author(s):  
Zhi-Ren Zhang ◽  
Shawn Zeltwanger ◽  
Stephen S. Smith ◽  
David C. Dawson ◽  
Nael A. McCarty
Keyword(s):  
Transmembrane Domain ◽  
Single Channel ◽  
Fold Increase ◽  
Open Probability ◽  
Whole Cell ◽  
Channel Current ◽  
Outward Rectification ◽  
Voltage Dependent ◽  
Excised Patches ◽  
Active Channels

A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current ( i)-voltage ( V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current ( I)- V relationship ( I = N × P o × i, where N is the number of active channels and P o is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the P o of V317E CFTR is voltage dependent. The decrease in P o at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P o- V relationship did not depend on the direction of Cl− movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.

A large conductance K(+)-selective channel of guinea pig villus enterocytes is Ca2+ independent

1992 ◽  
Vol 262 (2) ◽  
pp. G369-G374 ◽  
Author(s):  
G. M. Mintenig ◽  
A. S. Monaghan ◽  
F. V. Sepulveda
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
K Channel ◽  
Constant Field ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Current Voltage ◽  
Selective Channel ◽  
K Concentration ◽  
Current Voltage Relationship

The presence of K(+)-selective channels has been probed in enterocytes isolated from guinea pig small intestinal villi by the patch-clamp technique. A channel with a single-channel conductance of approximately 130 pS was observed in excised inside-out patches bathed in symmetrical K+. A change in the K+ concentration in the intracellular aspect of the membrane altered the current-voltage relationship as expected from the constant-field equation when it is assumed that K+ is the only permeant ion. A change in Cl- concentration was without effect. Neither the activity of the channel nor its conductance was altered by addition of ATP or Ba2+ to the intracellular side of the patches. Changes in the free Ca2+ concentration were also without effect. The channel's open probability showed no voltage dependence and appeared only occasionally active in cell-attached patches where it had a linear current-voltage relation. The K+ channel described, which cannot be readily classified in any of the known classes of K+ channels, might provide an exit pathway for K+ recycling in guinea pig villus enterocytes.

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