scholarly journals Three Types of Ion Channels in the Cell Membrane of Mouse Fibroblasts

2017 ◽  
pp. 63-73
Author(s):  
M. KOSELSKI ◽  
A. OLSZEWSKA ◽  
A. HORDYJEWSKA ◽  
T. MAŁECKA-MASSALSKA ◽  
K. TREBACZ
Keyword(s):  
Reversal Potential ◽  
Equilibrium Potential ◽  
Cation Channels ◽  
Anion Channels ◽  
Open Probability ◽  
K Channels ◽  
Nonselective Cation Channels ◽  
Inside Out ◽  
Small Conductance ◽  
High Conductance

Patch clamp recordings carried out in the inside-out configuration revealed activity of three kinds of channels: nonselective cation channels, small-conductance K+ channels, and large-conductance anion channels. The nonselective cation channels did not distinguish between Na+ and K+. The unitary conductance of these channels reached 28 pS in a symmetrical concentration of 200 mM NaCl. A lower value of this parameter was recorded for the small-conductance K+ channels and in a 50-fold gradient of K+ (200 mM/4 mM) it reached 8 pS. The high selectivity of these channels to potassium was confirmed by the reversal potential (-97 mV), whose value was close to the equilibrium potential for potassium (-100 mV). One of the features of the large-conductance anion channels was high conductance amounting to 493 pS in a symmetrical concentration of 200 mM NaCl. The channels exhibited three subconductance levels. Moreover, an increase in the open probability of the channels at voltages close to zero was observed. The anion selectivity of the channels was low, because the channels were permeable to both Cl- and gluconate – a large anion. Research on the calcium dependence revealed that internal calcium activates nonselective cation channels and small-conductance K+ channels, but not large-conductance anion channels.

scholarly journals Single channel characteristics of a high conductance anion channel in "sarcoballs".

1989 ◽  
Vol 93 (3) ◽  
pp. 385-410 ◽  
Author(s):  
G D Hals ◽  
P G Stein ◽  
P T Palade
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Surface Membrane ◽  
Reversal Potential ◽  
Anion Channel ◽  
Ion Concentration ◽  
Voltage Sensitivity ◽  
Anion Channels ◽  
Open Probability ◽  
High Conductance

Previously undescribed high conductance single anion channels from frog skeletal muscle sarcoplasmic reticulum (SR) were studied in native membrane using the "sarcoball" technique (Stein and Palade, 1988). Excised inside-out patches recorded in symmetrical 200 mM TrisCl show the conductance of the channel's predominant state was 505 +/- 25 pS (n = 35). From reversal potentials, the Pcl/PK ratio was 45. The slope conductance vs. Cl- ion concentration curve saturates at 617 pS, with K0.5 estimated at 77 mM. The steady-state open probability (Po) vs. holding potential relationship produces a bell-shaped curve, with Po values reaching a maximum near 1.0 at 0 mV, and falling off to 0.05 at +/- 25 mV. Kinetic analysis of the voltage dependence reveals that while open time constants are decreased somewhat by increases in potential, the largest effect is an increase in long closed times. Despite the channel's high conductance, it maintains a moderate selectivity for smaller anions, but will not pass larger anions such as gluconate, as determined by reversal-potential shifts. At least two substates different from the main open level are distinguishable. These properties are unlike those described for mitochondrial voltage-dependent anion channels or skeletal muscle surface membrane Cl channels and since SR Ca channels are present in equally high density in sarcoball patches, we propose these sarcoball anion channels originate from the SR. Preliminary experiments recording currents from frog SR anion channels fused into liposomes indicate that either biochemical isolation and/or alterations in lipid environment greatly decrease the channel's voltage sensitivity. These results help underline the potential significance of using sarcoballs to study SR channels. The steep voltage sensitivity of the sarcoball anion channel suggests that it could be more actively involved in the regulation of Ca2+ transport by the SR.

Regulation of high-conductance anion channels by G proteins and 5-HT1A receptors in CHO cells

1993 ◽  
Vol 264 (3) ◽  
pp. F490-F495 ◽  
Author(s):  
A. W. Mangel ◽  
J. R. Raymond ◽  
J. G. Fitz
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Single Channel ◽  
Chinese Hamster ◽  
Channel Activity ◽  
Anion Channels ◽  
Open Probability ◽  
Single Channel Currents ◽  
High Conductance

This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDP beta S, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.

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.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule

1995 ◽  
Vol 268 (4) ◽  
pp. F569-F580 ◽  
Author(s):  
L. C. Stoner ◽  
G. E. Morley
Keyword(s):  
Apical Membrane ◽  
Physiological Role ◽  
Reversal Potential ◽  
K Channel ◽  
Apical Surface ◽  
Open Probability ◽  
Volume Regulatory Decrease ◽  
Collecting Tubules ◽  
High Conductance

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P ' 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

Characterization of high-conductance anion channels in rat bile duct epithelial cells

1992 ◽  
Vol 262 (4) ◽  
pp. G703-G710 ◽  
Author(s):  
J. M. McGill ◽  
S. Basavappa ◽  
J. G. Fitz
Keyword(s):  
Bile Duct ◽  
Epithelial Cells ◽  
Equilibrium Potential ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Pipette Solution ◽  
Duct Ligation ◽  
Single Channel Currents ◽  
Rat Bile ◽  
High Conductance

We have utilized patch clamp recording techniques to identify a high-conductance anion channel in the plasma membrane of rat bile duct epithelial cells. Cells were isolated from the intrahepatic bile duct 2-6 wk after bile duct ligation. Channels were present in 27% (28/102) of excised patches, and, with 150 mM Cl- in bath and pipette solutions, the slope conductance of the fully open level was approximately 364 +/- 18 pS (n = 8) with current reversal = 0 +/- 1 mV. Channel characteristics were not affected by substitution of K+ for Na+ in the pipette solution; but substitution of HCO3-, gluconate, or increased NaCl caused a shift in the reversal potential toward the new equilibrium potential for Cl-. The permeability ratios were PHCO3-/PCl- = 0.51 +/- 0.03 (n = 5), Pgluconate/PCl- = 0.12 +/- 0.04 (n = 7), and PNa+/PCl- = 0.11 +/- 0.02 (n = 3). Current transitions to a subconductance level at 72% of the fully open level were present in most studies. Channel open probability was greatest near 0 mV and decreased rapidly outside of -20 to +20 mV because of voltage-dependent channel closure. The time course for current relaxation of summed single channel currents could be described by a single exponential with more rapid channel closure as the magnitude of the voltage step away from 0 mV increased. In the cell-attached configuration, the channel was rarely open (4/35, 11%) but opening could be induced by negative pipette pressure (5/14, 35%). Possible physiological roles for this channel are discussed.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

scholarly journals Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).

1992 ◽  
Vol 99 (6) ◽  
pp. 841-862 ◽  
Author(s):  
F Markwardt ◽  
G Isenberg
Keyword(s):  
Steady State ◽  
Time Course ◽  
Boltzmann Distribution ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
K Channels ◽  
The Mean ◽  
Inside Out

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.

Apical K+ conductance in maturing rabbit principal cell

1997 ◽  
Vol 272 (3) ◽  
pp. F397-F404 ◽  
Author(s):  
L. M. Satlin ◽  
L. G. Palmer
Keyword(s):  
Reversal Potential ◽  
Principal Cell ◽  
K Channel ◽  
Postnatal Life ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inward Currents ◽  
K Secretion

Net K+ secretion is not detected in cortical collecting ducts (CCDs) isolated from newborn rabbits and perfused in vitro. To establish whether a low apical K+ permeability of the neonatal principal cell limits K+ secretion early in life, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to study the properties and density of conducting K+ channels in principal cells. With KCl in the pipette and a NaCl solution warmed to 37 degrees C in the bath, inward currents with a conductance of approximately 42 pS were observed in 0% (0 out of 13 or 0/13), 10% (2/21), 18% (5/28), 29% (4/14), and 56% (10/18) of cell-attached patches obtained in 1-, 2-, 3-, 4-, and 5-wk-old animals, respectively. The conductance and reversal potential of this channel led us to suspect that it represented the low-conductance K+ channel previously described in the rat CCD by L. G. Palmer, L. Antonian, and G. Frindt (J. Gen. Physiol. 104: 693-710, 1994). The mean number of open channels per patch (NPo) increased progressively (P < 0.05) after birth, from 0 at 1 wk, to 0.06 +/- 0.04 at 2 wk, to 0.40 +/- 0.18 at 3 wk, to 0.74 +/- 0.41 at 4 wk, and to 1.06 +/- 0.28 at 5 wk. The increase in NPo appeared to be due primarily to a developmental increase in N, which is the number of channels; open probability, Po, remained constant at approximately 0.5 for all channels identified after the 2nd wk of life. The increase in number of conducting K+ channels during postnatal life is likely to contribute to the maturational increase in net K+ secretion in the CCDs.

Angiotensin II activation of Ca(2+)-permeant nonselective cation channels in rat adrenal glomerulosa cells

1996 ◽  
Vol 271 (5) ◽  
pp. C1705-C1715 ◽  
Author(s):  
D. P. Lotshaw ◽  
F. Li
Keyword(s):  
Angiotensin Ii ◽  
Channel Gating ◽  
Reversal Potential ◽  
Inward Rectification ◽  
Ang Ii ◽  
Permeability Ratio ◽  
Open Probability ◽  
Glomerulosa Cells ◽  
Single Channel Currents ◽  
Inside Out

A Ca(2+)-permeant, nonselective cation channel was observed in cell-attached and inside-out membrane patches from rat adrenal glomerulosa cells maintained in primary cell culture. In cell-attached patches under near physiological ionic conditions, single-channel currents exhibited a reversal potential near -10 mV, inward rectification, a nearly linear slope conductance between 0 and -80 mV of 17.4 pS, and voltage-dependent block at potentials more negative than -80 mV. Channels exhibiting identical conductance and gating properties were observed in inside-out patches; however, channel gating ran down within minutes in this configuation. In the inside-out configuration, channel gating did not require cytosolic Ca2+ (Ca2+ < 10(-9) M), and inward rectification was relieved by removal of intracellular Mg2+. Relative ionic permeability was calculated using reversal potential measurements from inside-out patches under bi-ionic conditions. The channel discriminated poorly among monovalent cations (PLi > PK > PCs > PNa) and was not significantly permeable to anions. The channel was permeable to Ca2+, exhibiting a relative permeability ratio of 0.29 PCa/PNa) when measured with 110 mM Ca2+ on the intracellular face and a permeability ratio of 4.38 (PCa/PNa) with 110 mM Ca2+ on the extracellular face. Channel gating behavior was episodic with open times ranging from milliseconds to tens of seconds and closed times lasting up to several minutes or longer. Channel gating appeared to be relatively voltage independent except that mean channel open time and open probability were reduced by membrane hyperpolarization. In cell-attached patches, bath application of 1 nM angiotensin II (ANG II) increased the channel open probability, primarily affecting channels exhibiting a low open probability, primarily affecting channels exhibiting a low open probability before stimulation. With the use of nystatin perforated-patch current clamp to measure membrane potential, ANG II was observed to induce large transient membrane depolarizations, consistent with activation of an inward current. We hypothesize that this channel is an important component of ANG II-induced membrane depolarization and Ca2+ influx during stimulation of aldosterone secretion.

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