scholarly journals Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH

2016 ◽  
Vol 148 (3) ◽  
pp. 213-226 ◽  
Author(s):  
Laurent Pinelli ◽  
Antoine Nissant ◽  
Aurélie Edwards ◽  
Stéphane Lourdel ◽  
Jacques Teulon ◽  
...  
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Minor Effect ◽  
Intercalated Cells ◽  
Distal Nephron ◽  
Open Probability ◽  
Cl Channels ◽  
Active Channels ◽  
Cl Conductance

ClC-K2, a member of the ClC family of Cl− channels and transporters, forms the major basolateral Cl− conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl− absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl−, and Ca2+ on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca2+ strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl− has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl−/HCO3− exchange in type B intercalated cells.

Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers

1989 ◽  
Vol 256 (4) ◽  
pp. C902-C912 ◽  
Author(s):  
R. J. Bridges ◽  
R. T. Worrell ◽  
R. A. Frizzell ◽  
D. J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Kinetic Scheme ◽  
Disulfonic Acid ◽  
Lipid Bilayer Membranes ◽  
Plasma Membrane Vesicles ◽  
Open Probability ◽  
Cl Channel ◽  
Cl Channels

We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.

Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

2007 ◽  
Vol 293 (1) ◽  
pp. F236-F244 ◽  
Author(s):  
Ling Yu ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Heavy Metal ◽  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Epithelial Cell Line ◽  
Open Probability ◽  
Renal Epithelial Cell ◽  
Histidine Residues ◽  
Voltage Dependent ◽  
Renal Epithelial Cell Line

To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels ( N) × open probability ( Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

Psoralens: novel modulators of Cl- secretion

1997 ◽  
Vol 272 (3) ◽  
pp. C976-C988 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. J. Bridges ◽  
R. A. Frizzell
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Receptor Activation ◽  
Sustained Response ◽  
Rat Colon ◽  
Cl Secretion ◽  
Cl Channel ◽  
Cl Channels ◽  
Cl Conductance

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

scholarly journals Shaker potassium channel gating. II: Transitions in the activation pathway.

1994 ◽  
Vol 103 (2) ◽  
pp. 279-319 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
J Dittman ◽  
R W Aldrich
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ionic Current ◽  
Activation Process ◽  
Charge Movement ◽  
Open Probability ◽  
Current Time ◽  
Gating Charge

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

scholarly journals Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage

2016 ◽  
Vol 310 (4) ◽  
pp. F311-F321 ◽  
Author(s):  
Oleg Zaika ◽  
Oleg Palygin ◽  
Viktor Tomilin ◽  
Mykola Mamenko ◽  
Alexander Staruschenko ◽  
...  
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Sodium Reabsorption ◽  
Cortical Collecting Duct ◽  
Open Probability ◽  
Whole Cell ◽  
Principal Cells ◽  
Basolateral Side ◽  
Kinase Cascade

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K+-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na+-K+-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K+-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na+ reabsorption in the CCD.

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.

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.

Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells

2007 ◽  
Vol 293 (5) ◽  
pp. F1666-F1677 ◽  
Author(s):  
Ling Yu ◽  
Hui-Fang Bao ◽  
Julie L. Self ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Single Channel ◽  
Na Channel ◽  
Dependent Manner ◽  
Channel Measurements ◽  
Distal Nephron ◽  
Open Probability ◽  
A6 Cells ◽  
A Cell

Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2−) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 μM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2−(with the O2−scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 ± 0.03 to 0.03 ± 0.01. We also found that increasing endogenous O2−in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 ± 0.05) after superoxide dismutase and 1.5 μM NO coincubation (0.21 ± 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2−by ∼30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2−alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.

scholarly journals Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels

2004 ◽  
Vol 124 (5) ◽  
pp. 463-474 ◽  
Author(s):  
Tommaso Fellin ◽  
Siro Luvisetto ◽  
Michele Spagnolo ◽  
Daniela Pietrobon
Keyword(s):  
Calcium Channels ◽  
Time Scale ◽  
Neuronal Excitability ◽  
Voltage Dependence ◽  
Single Channel ◽  
Preceding Paper ◽  
Hek293 Cells ◽  
Open Probability ◽  
Modal Gating ◽  
Complex Modal

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445–461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.

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