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 Transfer of twelve charges is needed to open skeletal muscle Na+ channels.

1995 ◽  
Vol 106 (6) ◽  
pp. 1053-1068 ◽  
Author(s):  
B Hirschberg ◽  
A Rovner ◽  
M Lieberman ◽  
J Patlak
Keyword(s):  
Skeletal Muscle ◽  
Membrane Potential ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Fast Inactivation ◽  
Open Probability ◽  
Single Channel Analysis ◽  
Voltage Dependent ◽  
Function Models

Voltage-dependent Na+ channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single-channel recording to measure skeletal muscle Na+ channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, we have generated a clone of the rat skeletal muscle Na+ channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of S4 mutagenesis studies and structure/function models of the Na+ channel.

Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides

2006 ◽  
Vol 290 (5) ◽  
pp. F949-F957 ◽  
Author(s):  
Oleh Pochynyuk ◽  
Qiusheng Tong ◽  
Alexander Staruschenko ◽  
He-Ping Ma ◽  
James D. Stockand
Keyword(s):  
Binding Site ◽  
Transmembrane Domain ◽  
Second Messengers ◽  
Proximal Region ◽  
Signaling Cascades ◽  
Na Channel ◽  
Channel Activity ◽  
Open Probability ◽  
End Effector ◽  
Epithelial Na Channel

The epithelial Na+ channel (ENaC) is an end-effector of diverse cellular signaling cascades, including those with phosphatidylinositide second messengers. Recent evidence also suggests that in some instances, phospatidylinositides can directly interact with ENaC to increase channel activity by increasing channel open probability and/or membrane localization. We review here findings relevant to regulation of ENaC by phosphatidylinositol 4,5- bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3). Similar to its actions on other ion channels, PIP2 is permissive for ENaC openings having a direct effect on gating. The PIP2 binding site in ENaC involved in this regulation is most likely localized to the NH2 terminus of β-ENaC. PIP3 also affects ENaC gating but, rather than being permissive, augments open probability. The PIP3 binding site in ENaC involved in this regulation is localized to the proximal region of the COOH terminus of γ-ENaC just following the second transmembrane domain. In complementary pathways, PIP3 also impacts ENaC membrane levels through both direct actions on the channel and via a signaling cascade involving phosphoinositide 3-OH kinase (PI3-K) and the aldosterone-induced gene product serum and glucocorticoid-inducible kinase. The putative PIP3 binding site in ENaC involved in direct regulation of channel membrane levels has not yet been identified.

scholarly journals The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na+channel (ENaC): IUPHAR Review 19

2016 ◽  
Vol 173 (18) ◽  
pp. 2671-2701 ◽  
Author(s):  
Emilie Boscardin ◽  
Omar Alijevic ◽  
Edith Hummler ◽  
Simona Frateschi ◽  
Stephan Kellenberger
Keyword(s):  
Ion Channels ◽  
Na Channel ◽  
Acid Sensing Ion Channels ◽  
Epithelial Na Channel

scholarly journals Regulation of the Epithelial Na+ Channel by Membrane Tension

1998 ◽  
Vol 112 (2) ◽  
pp. 97-111 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Muthangi Subramanyam
Keyword(s):  
Cytochalasin B ◽  
Single Channel ◽  
Direct Injection ◽  
Expression System ◽  
Cell Swelling ◽  
Na Channel ◽  
Bathing Solution ◽  
Membrane Tension ◽  
Whole Cell ◽  
Epithelial Na Channel

The sensitivity of αβγ rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers

1995 ◽  
Vol 268 (6) ◽  
pp. C1450-C1459 ◽  
Author(s):  
M. S. Awayda ◽  
I. I. Ismailov ◽  
B. K. Berdiev ◽  
D. J. Benos
Keyword(s):  
Hydrostatic Pressure ◽  
Pressure Gradient ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Stretch Activation ◽  
Hydrostatic Pressure Gradient ◽  
Epithelial Na Channel

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA 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.

scholarly journals Elementary mechanisms of calmodulin regulation of NaV1.5 producing divergent arrhythmogenic phenotypes

2021 ◽  
Vol 118 (21) ◽  
pp. e2025085118
Author(s):  
Po Wei Kang ◽  
Nourdine Chakouri ◽  
Johanna Diaz ◽  
Gordon F. Tomaselli ◽  
David T. Yue ◽  
...  
Keyword(s):  
Single Channel ◽  
Loss Of Function ◽  
Na Current ◽  
Open Probability ◽  
Atomic Structures ◽  
Single Channel Analysis ◽  
Inactivation Mechanism ◽  
Life Threatening ◽  
Channel Analysis ◽  
Cam Regulation

In cardiomyocytes, NaV1.5 channels mediate initiation and fast propagation of action potentials. The Ca2+-binding protein calmodulin (CaM) serves as a de facto subunit of NaV1.5. Genetic studies and atomic structures suggest that this interaction is pathophysiologically critical, as human mutations within the NaV1.5 carboxy-terminus that disrupt CaM binding are linked to distinct forms of life-threatening arrhythmias, including long QT syndrome 3, a “gain-of-function” defect, and Brugada syndrome, a “loss-of-function” phenotype. Yet, how a common disruption in CaM binding engenders divergent effects on NaV1.5 gating is not fully understood, though vital for elucidating arrhythmogenic mechanisms and for developing new therapies. Here, using extensive single-channel analysis, we find that the disruption of Ca2+-free CaM preassociation with NaV1.5 exerts two disparate effects: 1) a decrease in the peak open probability and 2) an increase in persistent NaV openings. Mechanistically, these effects arise from a CaM-dependent switch in the NaV inactivation mechanism. Specifically, CaM-bound channels preferentially inactivate from the open state, while those devoid of CaM exhibit enhanced closed-state inactivation. Further enriching this scheme, for certain mutant NaV1.5, local Ca2+ fluctuations elicit a rapid recruitment of CaM that reverses the increase in persistent Na current, a factor that may promote beat-to-beat variability in late Na current. In all, these findings identify the elementary mechanism of CaM regulation of NaV1.5 and, in so doing, unravel a noncanonical role for CaM in tuning ion channel gating. Furthermore, our results furnish an in-depth molecular framework for understanding complex arrhythmogenic phenotypes of NaV1.5 channelopathies.

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.

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