Ca2+-activated K+ channel inhibition by reactive oxygen species

2002 ◽  
Vol 282 (3) ◽  
pp. C461-C471 ◽  
Author(s):  
Marco A. Soto ◽  
Carlos González ◽  
Eduardo Lissi ◽  
Cecilia Vergara ◽  
Ramón Latorre
Keyword(s):  
Noise Analysis ◽  
K Channel ◽  
Amino Acid Residues ◽  
Channel Activity ◽  
Open Probability ◽  
Α Subunit ◽  
Channel Inhibition ◽  
Voltage Dependent ◽  
Active Channels ◽  
Extracellular Side

We studied the effect of H2O2 on the gating behavior of large-conductance Ca2+-sensitive voltage-dependent K+ (KV,Ca) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevisoocytes membranes expressing the human Slowpoke(h Slo) α-subunit. Exposure of the intracellular side of KV,Ca channels to H2O2 (4–23 mM) leads to a time-dependent decrease of the open probability ( P o) without affecting the unitary conductance. H2O2 did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H2O2 action. Desferrioxamine (60 μM) and cysteine (1 mM) completely inhibited the effect of H2O2, indicating that the decrease in P o was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H2O2. However, DTT did completely reverse the decrease in P o induced by the oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of KV,Ca channel activity promoted by DTT suggests that H2O2 treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing h Slo channels showed that H2O2 induced a decrease in current mediated by a decrease both in the number of active channels and P o.

scholarly journals Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP

1998 ◽  
Vol 112 (3) ◽  
pp. 333-349 ◽  
Author(s):  
Stefan Trapp ◽  
Peter Proks ◽  
Stephen J. Tucker ◽  
Frances M. Ashcroft
Keyword(s):  
Transmembrane Domain ◽  
K Channel ◽  
Channel Activity ◽  
Inhibitory Effects ◽  
Open Probability ◽  
Regulatory Subunits ◽  
Closed State ◽  
Channel Inhibition ◽  
Apparent Decrease ◽  
Atp Inhibition

The β cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2ΔC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2ΔC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2ΔC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 μM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

Maxi K+ channel in apical membrane of vestibular dark cells

1992 ◽  
Vol 262 (6) ◽  
pp. C1430-C1436 ◽  
Author(s):  
S. Takeuchi ◽  
D. C. Marcus ◽  
P. Wangemann
Keyword(s):  
Apical Membrane ◽  
K Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Dark Cells ◽  
Voltage Dependent ◽  
Cytosolic Side ◽  
Selective Channel ◽  
Extracellular Side ◽  
Vestibular Dark Cells

Recordings were made on excised apical membrane patches from vestibular dark cells from the semicircular canal of gerbils to determine if ion channels could be involved in the process of K+ secretion. Both nonselective cation channels [Am. J. Physiol. 262 (Cell Physiol. 31): C1430-C1436, 1992] and K(+)-selective channels were found. The K+ channels occurred in only 0.7% of the patches. In symmetrical 145 mM KCl solutions, the current-voltage (I-V) relation of the K(+)-selective channel was linear, indicating the absence of rectification, and the conductance was 240 +/- 8 pS (n = 8). The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relation in asymmetrical K+ and Na+ solutions and yielded a K+ permeability of 5.78 x 10(-13) cm3/s (n = 12). The channel was shown to be impermeable to Li+, NH4+, N-methyl-D-glucamine, and Cl-. Channel activity increased with depolarization and with increasing free [Ca2+]; for voltages between +40 and -60 mV, the strongest regulation occurred in the range 10(-6) to 10(-5) M Ca2+. Tetraethylammonium (2 x 10(-2) M) had from the cytosolic side no effect on the open probability (Po) but completely inhibited activity from the extracellular side. Po was reduced by Ba2+ (5 x 10(-3) M), verapamil (10(-4) M), quinine (10(-4) M), and quinidine (10(-4) and 10(-3) M), while lidocaine (5 x 10(-3) M) had no measurable effect on Po but decreased the amplitude. Rb+ and Cs+ were either poorly permeable or partially blocked the channel in a voltage-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Slo3 K+ Channels: Voltage and pH Dependence of Macroscopic Currents

2006 ◽  
Vol 128 (3) ◽  
pp. 317-336 ◽  
Author(s):  
Xue Zhang ◽  
Xuhui Zeng ◽  
Christopher J. Lingle
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ph Dependence ◽  
K Channel ◽  
Open Probability ◽  
Cytosolic Ph ◽  
Α Subunit ◽  
K Channels ◽  
C Terminus ◽  
Voltage Dependent

The mouse Slo3 gene (KCNMA3) encodes a K+ channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca2+ and voltage-activated BK-type channel, the Slo3 α subunit contains a pore module with homology to voltage-gated K+ channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K+ channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich–type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (−300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (zL) of the Slo3 closed–open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

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.

Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6)

1994 ◽  
Vol 267 (5) ◽  
pp. C1414-C1425 ◽  
Author(s):  
K. E. Kokko ◽  
P. S. Matsumoto ◽  
B. N. Ling ◽  
D. C. Eaton
Keyword(s):  
Prostaglandin E2 ◽  
Apical Cell ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
Channel Inhibition ◽  
Camp Levels

We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.

scholarly journals BK channel inhibition by strong extracellular acidification

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Yu Zhou ◽  
Xiao-Ming Xia ◽  
Christopher J Lingle
Keyword(s):  
Bk Channels ◽  
Bk Channel ◽  
Extracellular Acidification ◽  
Channel Inhibition ◽  
Wide Range ◽  
The Family ◽  
Voltage Dependent ◽  
Intracellular Organelles ◽  
Extracellular Side ◽  
Key Residues

Mammalian BK-type voltage- and Ca2+-dependent K+ channels are found in a wide range of cells and intracellular organelles. Among different loci, the composition of the extracellular microenvironment, including pH, may differ substantially. For example, it has been reported that BK channels are expressed in lysosomes with their extracellular side facing the strongly acidified lysosomal lumen (pH ~4.5). Here we show that BK activation is strongly and reversibly inhibited by extracellular H+, with its conductance-voltage relationship shifted by more than +100 mV at pHO 4. Our results reveal that this inhibition is mainly caused by H+ inhibition of BK voltage-sensor (VSD) activation through three acidic residues on the extracellular side of BK VSD. Given that these key residues (D133, D147, D153) are highly conserved among members in the voltage-dependent cation channel superfamily, the mechanism underlying BK inhibition by extracellular acidification might also be applicable to other members in the family.

Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule

1993 ◽  
Vol 264 (4) ◽  
pp. F760-F764 ◽  
Author(s):  
A. M. Hurst ◽  
J. S. Beck ◽  
R. Laprade ◽  
J. Y. Lapointe
Keyword(s):  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
K Channel ◽  
Bathing Solution ◽  
Channel Activity ◽  
Open Probability ◽  
Functional Link ◽  
Tightly Coupled ◽  
Stimulation Of

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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