Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane

2012 ◽  
Vol 302 (6) ◽  
pp. C868-C879 ◽  
Author(s):  
Diana Wesch ◽  
Mike Althaus ◽  
Pablo Miranda ◽  
Ignacio Cruz-Muros ◽  
Martin Fronius ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Channel ◽  
Hek293 Cells ◽  
Na Channel ◽  
Open Probability ◽  
Splice Isoforms ◽  
N Terminus ◽  
Lysine Residues ◽  
Tight Epithelia ◽  
Py Motif

The epithelial Na+ channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na+ reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, β, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ1 and δ2 , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ1 or δ2 , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ1 in Xenopus oocytes and HEK293 cells produces higher current levels than δ2 . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of β- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ2-N terminus. Analysis of δ2-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ1 higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αβγ-channels.

scholarly journals Endogenous Protease Activation of ENaC

2005 ◽  
Vol 126 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Adedotun Adebamiro ◽  
Yi Cheng ◽  
John P. Johnson ◽  
Robert J. Bridges
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Open Channels ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Protease Inhibition ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Channel Properties

Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain INa at some minimal threshold value.

scholarly journals Sodium channel subconductance levels measured with a new variance-mean analysis.

1988 ◽  
Vol 92 (4) ◽  
pp. 413-430 ◽  
Author(s):  
J B Patlak
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Open Time ◽  
Flexor Digitorum Brevis ◽  
Dissociated Cells ◽  
A New Technique ◽  
Flexor Digitorum

The currents through single Na+ channels were recorded from dissociated cells of the flexor digitorum brevis muscle of the mouse. At 15 degrees C the prolonged bursts of Na+ channel openings produced by application of the drug DPI 201-106 had brief sojourns to subconductance levels. The subconductance events were relatively rare and brief, but could be identified using a new technique that sorts amplitude estimates based on their variance. The resulting "levels histogram" had a resolution of the conductance levels during channel activity that was superior to that of standard amplitude histograms. Cooling the preparation to 0 degrees C prolonged the subconductance events, and permitted further quantitative analysis of their amplitudes, as well as clear observations of single-channel subconductance events from untreated Na+ channels. In all cases the results were similar: a subconductance level, with an amplitude of roughly 35% of the fully open conductance and similar reversal potential, was present in both drug-treated and normal Na+ channels. Drug-treated channels spent approximately 3-6% of their total open time in the subconductance state over a range of potentials that caused the open probability to vary between 0.1 and 0.9. The summed levels histograms from many channels had a distinctive form, with broader, asymmetrical open and substate distributions compared with those of the closed state. Individual subconductance events to levels other than the most common 35% were also observed. I conclude that subconductance events are a normal subset of the open state of Na+ channels, whether or not they are drug treated. The subconductance events may represent a conformational alteration of the channel that occurs when it conducts ions.

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells

1995 ◽  
Vol 269 (3) ◽  
pp. C733-C738 ◽  
Author(s):  
L. Vaca ◽  
D. L. Kunze
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Single Channel ◽  
Vascular Endothelial Cells ◽  
Channel Activity ◽  
Open Probability ◽  
Aortic Endothelial Cells ◽  
Control Value ◽  
Selective Channel ◽  
Inside Out

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

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.

Effects of vasopressin and cAMP on single amiloride-blockable Na channels

1991 ◽  
Vol 260 (5) ◽  
pp. C1071-C1084 ◽  
Author(s):  
Y. Marunaka ◽  
D. C. Eaton
Keyword(s):  
Sodium Channels ◽  
Arginine Vasopressin ◽  
Na Channel ◽  
Frequency Of Occurrence ◽  
Na Channels ◽  
Distal Nephron ◽  
Open Probability ◽  
Tight Epithelia ◽  
Two Peaks ◽  
Individual Patch

To determine the mechanism by which vasopressin increases sodium transport in sodium-transporting, tight epithelia, we examined single amiloride-blockable Na channels in membrane patches from cultured distal nephron cells (A6) either before or after treatment with arginine vasopressin. Pretreatment of cells with vasopressin (40 mU/ml) for 40-50 min increases NPo (N, the number of Na channels; Po, the open probability of an individual Na channel). The increase in NPo is due to an increase in the number of conductive Na channels with little or no change in the open probability of individual Na channels. Pretreatment of cells for 1 h with 1 mM N6,2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (DBcAMP) also increased NPo. The increase in NPo caused by DBcAMP pretreatment is also due to the increase in the number of conductive Na channels with no change in the open probability of individual Na channels. Cells pretreated with cholera toxin (CTX; 250 ng/ml) for 4 h appeared similar to cells that had been treated with vasopressin or DBcAMP; that is, the number of Na channels per patch increased with little or no effect on the open probability of individual Na channels. For patches from many untreated cells, when the frequency of occurrence is plotted against the number of channels in an individual patch, the histogram consists of a single peak with a number of channels per patch of 2.0 +/- 1.5 (+/- SD, 126 patches). After pretreatment of cells with vasopressin, DBcAMP, or CTX, the same histogram contains two peaks after vasopressin of 1.8 +/- 1.2 and 9.2 +/- 1.5 (+/- SD, 38 and 53 patches, respectively). These observations suggest that pretreatment of cells with vasopressin, DBcAMP, or CTX may act by promoting insertion of clusters of new sodium channels.

scholarly journals SAP97 regulates Kir2.3 channels by multiple mechanisms

2009 ◽  
Vol 297 (4) ◽  
pp. H1387-H1397 ◽  
Author(s):  
Karen L. Vikstrom ◽  
Ravi Vaidyanathan ◽  
Susan Levinsohn ◽  
Ryan P. O'Connell ◽  
Yueming Qian ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Channel ◽  
Hek293 Cells ◽  
Scaffolding Protein ◽  
Cell Surface Expression ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Whole Cell ◽  
The Impact

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ∼13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.

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 Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies.

1992 ◽  
Vol 172 (1) ◽  
pp. 271-287
Author(s):  
A Bertl ◽  
C L Slayman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Single Channel ◽  
K Channel ◽  
Substrate Uptake ◽  
Alkaline Ph ◽  
Open Probability ◽  
Extracellular Medium ◽  
Yeast Saccharomyces Cerevisiae ◽  
Charge Balancing

Detailed patch-clamp studies have been made of ion channels in the plasma membrane and tonoplast of the yeast Saccharomyces cerevisiae. The predominant tonoplast channel is a high-conductance cation-selective inward rectifier (passing ions easily into the cytoplasm from the vacuole), with its open probability (Po) peaking at about -80 mV (cytoplasm negative) and falling to near zero at +80 mV. It has a maximal slope conductance of approximately 150 pS in 100 mmol l-1 KCl, and conducts Na+, K+ and Ca2+. Elevated cytoplasmic Ca2+ concentration, alkaline pH and reducing agents can activate the channel, its likely physiological function being to adjust cytoplasmic Ca2+ concentration from the vacuolar reservoir. The predominant plasma-membrane channel is a strongly outward rectifying K+ channel (passing K+ easily out of the cytoplasm to the extracellular medium), which is activated by positive-going membrane voltages as well as by elevated cytoplasmic Ca2+ concentration and alkaline pH. Interaction between membrane voltage and [Ca2+]cyt is complex and defines three parallel closed states for the channel: a Ca(2+)-independent brief closure (I), a calcium-inhibited long closure (G) and, at large positive voltages, a calcium-induced brief blockade (B). This channel is likely to function in steady-state turgor regulation and in charge balancing during proton-coupled substrate uptake.

scholarly journals Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel

2007 ◽  
Vol 130 (4) ◽  
pp. 399-413 ◽  
Author(s):  
Oleh Pochynyuk ◽  
Qiusheng Tong ◽  
Jorge Medina ◽  
Alain Vandewalle ◽  
Alexander Staruschenko ◽  
...  
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Second Messengers ◽  
Transmembrane Domains ◽  
Effector Proteins ◽  
Na Channel ◽  
Open Probability ◽  
Single Channel Analysis ◽  
Molecular Determinants ◽  
Epithelial Na Channel

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

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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