Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1036-C1044 ◽  
Author(s):  
Z. Fan ◽  
Y. Tokuyama ◽  
J. C. Makielski
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Activation Effect ◽  
Insulin Secreting Cells ◽  
Single Channel Currents

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

1998 ◽  
Vol 275 (2) ◽  
pp. C535-C543 ◽  
Author(s):  
C. A. Obejero-Paz ◽  
M. Auslender ◽  
A. Scarpa
Keyword(s):  
Okadaic Acid ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell ◽  
A7r5 Cells ◽  
High Level ◽  
Single Channel Currents ◽  
Channel Currents

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

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.

Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels

1998 ◽  
Vol 274 (4) ◽  
pp. L475-L484 ◽  
Author(s):  
Lucky Jain ◽  
Xi-Juan Chen ◽  
Lou Ann Brown ◽  
Douglas C. Eaton
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Cation Channel ◽  
Alveolar Type ◽  
Apical Surface ◽  
Type Ii ◽  
Patch Clamp Technique ◽  
Open Probability

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( P o), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the P o by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; P o, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; −42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( P o, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.

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.

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.

Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry

1993 ◽  
Vol 264 (3) ◽  
pp. F565-F574 ◽  
Author(s):  
G. Frindt ◽  
R. B. Silver ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Homeostasis ◽  
Feedback Regulation ◽  
Feedback Mechanism ◽  
Acute Administration ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Single Channel Currents

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and single-channel currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of amiloride (10 microM) or benzamil (0.5 microM) to the superfusate resulted in a twofold increase in the mean number of open channels (NPo) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NPo reversed within 3 min after removal of amiloride. The increase in NPo was accounted for, at least in part, by an increase in Po. Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NPo. No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel Po by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Electrophysiological Properties of Mouse Horizontal Cell GABAA Receptors

2004 ◽  
Vol 92 (5) ◽  
pp. 2789-2801 ◽  
Author(s):  
Andreas Feigenspan ◽  
Reto Weiler
Keyword(s):  
Single Channel ◽  
Horizontal Cell ◽  
Chloride Ions ◽  
Horizontal Cells ◽  
Hill Coefficient ◽  
Mouse Retina ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Steady State Current ◽  
Single Channel Currents

GABA-induced currents have been characterized in isolated horizontal cells from lower vertebrates but not in mammalian horizontal cells. Therefore horizontal cells were isolated after enzymatical and mechanical dissociation of the adult mouse retina and visually identified. We recorded from horizontal cell bodies using the whole cell and outside-out configuration of the patch-clamp technique. Extracellular application of GABA induced inward currents carried by chloride ions. GABA-evoked currents were completely and reversibly blocked by the competitive GABAA receptor antagonist bicuculline (IC50 = 1.7 μM), indicating expression of GABAA but not GABAC receptors. Their affinity for GABA was moderate (EC50 = 30 μM), and the Hill coefficient was 1.3, corresponding to two GABA binding sites. GABA responses were partially reduced by picrotoxin with differential effects on peak and steady-state current values. Zinc blocked the GABA response with an IC50 value of 7.3 μM in a noncompetitive manner. Furthermore, GABA receptors of horizontal cells were modulated by extracellular application of diazepam, zolpidem, methyl 6,7-dimethoxy-4-ethyl-β-carboxylate, pentobarbital, and alphaxalone, thus showing typical pharmacological properties of CNS GABAA receptors. GABA-evoked single-channel currents were characterized by a main conductance state of 29.8 pS and two subconductance states (20.2 and 10.8 pS, respectively). Kinetic analysis of single-channel events within bursts revealed similar mean open and closed times for the main conductance and the 20.2-pS subconductance state, resulting in open probabilities of 44.6 and 42.7%, respectively. The ratio of open to closed times, however, was significantly different for the 10.8-pS subconductance state with an open probability of 57.2%.

Peroxynitrite reversibly inhibits Ca2+-activated K+ channels in rat cerebral artery smooth muscle cells

2000 ◽  
Vol 278 (6) ◽  
pp. H1883-H1890 ◽  
Author(s):  
Anna K. Brzezinska ◽  
Debebe Gebremedhin ◽  
William M. Chilian ◽  
Balaraman Kalyanaraman ◽  
Stephen J. Elliott
Keyword(s):  
Single Channel ◽  
Cerebral Arteries ◽  
Ionic Current ◽  
Inhibitory Effect ◽  
Bk Channels ◽  
Bk Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Cell Current

Peroxynitrite (ONOO−) is a contractile agonist of rat middle cerebral arteries. To determine the mechanism responsible for this component of ONOO−bioactivity, the present study examined the effect of ONOO− on ionic current and channel activity in rat cerebral arteries. Whole cell recordings of voltage-clamped cells were made under conditions designed to optimize K+ current. The effects of iberiotoxin, a selective inhibitor of large-conductance Ca2+-activated K+ (BK) channels, and ONOO− (10–100 μM) were determined. At a pipette potential of +50 mV, ONOO− inhibited 39% of iberiotoxin-sensitive current. ONOO− was selective for iberiotoxin-sensitive current, whereas decomposed ONOO− had no effect. In excised, inside-out membrane patches, channel activity was recorded using symmetrical K+solutions. Unitary currents were sensitive to increases in internal Ca2+ concentration, consistent with activity due to BK channels. Internal ONOO− dose dependently inhibited channel activity by decreasing open probability and mean open times. The inhibitory effect of ONOO− could be overcome by reduced glutathione. Glutathione, added after ONOO−, restored whole cell current amplitude to control levels and reverted single-channel gating to control behavior. The inhibitory effect of ONOO− on membrane K+ current is consistent with its contractile effects in isolated cerebral arteries and single myocytes. Taken together, our data suggest that ONOO− has the potential to alter cerebral vascular tone by inhibiting BK channel activity.

Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells

2013 ◽  
Vol 304 (11) ◽  
pp. H1415-H1427 ◽  
Author(s):  
Piotr Bednarczyk ◽  
Agnieszka Koziel ◽  
Wieslawa Jarmuszkiewicz ◽  
Adam Szewczyk
Keyword(s):  
Potassium Channel ◽  
Mitochondrial Membrane ◽  
Single Channel ◽  
Dependent Manner ◽  
Channel Activity ◽  
Inner Mitochondrial Membrane ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Α Subunit ◽  
Ea.Hy926 Cells

In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

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