scholarly journals Allosteric modulation of alternatively spliced Ca2+-activated Cl−channels TMEM16A by PI(4,5)P2and CaMKII

2020 ◽  
Vol 117 (48) ◽  
pp. 30787-30798
Author(s):  
Woori Ko ◽  
Seung-Ryoung Jung ◽  
Kwon-Woo Kim ◽  
Jun-Hee Yeon ◽  
Cheon-Gyu Park ◽  
...  
Keyword(s):  
Binding Site ◽  
In Silico ◽  
Single Channel ◽  
Splice Variants ◽  
Noise Analysis ◽  
Mechanistic Explanation ◽  
Intracellular Loop ◽  
Open Probability ◽  
In Silico Simulation ◽  
Alternatively Spliced

Transmembrane 16A (TMEM16A, anoctamin1), 1 of 10 TMEM16 family proteins, is a Cl−channel activated by intracellular Ca2+and membrane voltage. This channel is also regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. We find that two splice variants of TMEM16A show different sensitivity to endogenous PI(4,5)P2degradation, where TMEM16A(ac) displays higher channel activity and more current inhibition by PI(4,5)P2depletion than TMEM16A(a). These two channel isoforms differ in the alternative splicing of the c-segment (exon 13). The current amplitude and PI(4,5)P2sensitivity of both TMEM16A(ac) and (a) are significantly strengthened by decreased free cytosolic ATP and by conditions that decrease phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII). Noise analysis suggests that the augmentation of currents is due to a rise of single-channel current (i), but not of channel number (N) or open probability (PO). Mutagenesis points to arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and to serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the action of PI(4,5)P2. In silico simulation suggests how phosphorylation of S673 allosterically and differently changes the structure of the distant PI(4,5)P2-binding site between channel splice variants with and without the c-segment exon. In sum, our study reveals the following: differential regulation of alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in silico simulation.

scholarly journals Interactions of a Reversible Ryanoid (21-Amino-9α-Hydroxy-Ryanodine) with Single Sheep Cardiac Ryanodine Receptor Channels

1998 ◽  
Vol 112 (1) ◽  
pp. 55-69 ◽  
Author(s):  
Bhavna Tanna ◽  
William Welch ◽  
Luc Ruest ◽  
John L. Sutko ◽  
Alan J. Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Single Channel ◽  
Single Channels ◽  
Open Probability ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Channel Protein ◽  
High Affinity ◽  
Conductance State

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels

2001 ◽  
Vol 280 (5) ◽  
pp. C1130-C1139 ◽  
Author(s):  
Jichang Li ◽  
Ana M. Correa
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Noise Analysis ◽  
Single Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Slowing Down ◽  
Conductance Increase

Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (∼17%) and maximum open probability (∼20%). Single-channel conductance was larger (∼20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.

Steroid hormone-dependent expression of blockersensitive ENaCs in apical membranes of A6 epithelia

1997 ◽  
Vol 273 (5) ◽  
pp. C1650-C1656 ◽  
Author(s):  
Lynn M. Baxendale-Cox ◽  
Randall L. Duncan ◽  
Xuehong Liu ◽  
Kieron Baldwin ◽  
Willem J. Els ◽  
...  
Keyword(s):  
Growth Medium ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Noise Analysis ◽  
Short Circuit Current ◽  
Growth Media ◽  
Open Probability ◽  
A6 Epithelia ◽  
Single Channel Currents

Weak channel blocker-induced noise analysis was used to determine the way in which the steroids aldosterone and corticosterone stimulated apical membrane Na+ entry into the cells of tissue-cultured A6 epithelia. Among groups of tissues grown on a variety of substrates, in a variety of growth media, and with cells at passages 73–112, the steroids stimulated both amiloride-sensitive and amiloride-insensitive Na+ transport as measured by short-circuit currents in chambers perfused with either growth medium or a Ringer solution. From baseline rates of blocker-sensitive short-circuit current between 2 and 7 μA/cm2, transport was stimulated about threefold in all groups of experiments. Single channel currents averaged near 0.3 pA (growth medium) and 0.5 pA (Ringer) and were decreased 6–20% from controls by steroid due to the expected decreases of fractional transcellular resistance. Irrespective of baseline transport rates, the steroids in all groups of tissues stimulated transport by increase of the density of blocker-sensitive epithelial Na+ channels (ENaCs). Channel open probability was the same in control and stimulated tissues, averaging ∼0.3 in all groups of tissues. Accordingly, steroid-mediated increases of open channel density responsible for stimulation of Na+ transport are due to increases of the apical membrane pool of functional channels and not their open probability.

scholarly journals CFTR displays voltage dependence and two gating modes during stimulation.

1994 ◽  
Vol 104 (3) ◽  
pp. 541-566 ◽  
Author(s):  
H Fischer ◽  
T E Machen
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Current Noise ◽  
Corner Frequency ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Closed State ◽  
Open States ◽  
Multichannel Recordings ◽  
Long Channel

The patch-clamp technique in conjunction with current noise analysis was employed to clarify the events underlying the regulation of the CFTR (cystic fibrosis transmembrane conductance regulator) during cAMP-dependent stimulation. 3T3 fibroblast cells expressing the CFTR were stimulated in cell-attached mode with forskolin. The number (N) of activated channels per patch ranged from 1 to approximately 100. In true single-channel recordings, CFTR's gating was best described by two open states (approximately 5 and approximately 100 ms) and three closed states (< or = 5, approximately 100, and approximately 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and approximately 50 Hz, respectively. Single-channel time constants were dependent on voltage. The fastest closed state increased its contribution from 48% at +100 mV to 87% at -100 mV, and the medium open state reduced its length to one half, resulting in gating dominated by fast events. Similarly, the fast Lorentzian increased its amplitude, and its corner frequency increased from 44 Hz at +100 mV to 91 Hz at -100 mV, while the slow Lorentzian was voltage independent. In multi-channel recordings N.Po (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased Po of CFTR to approximately 0.5, which resulted from a decrease of the longest closed state while the faster open and closed states were unaffected. Neither corner frequency was affected during stimulation. Recordings from multichannel patches revealed in addition, unique, very long channel openings (high Po mode, average 13 s). Channels exhibiting high Po (i.e., Po approximately 1.0) or low Po (i.e., Po approximately 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high Po mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 microM) favored transition into the high Po mode, suggesting a cellularly mediated regulation of model switching due to a fundamental change in configuration of the CFTR. Thus, during stimulation the CFTR increased its activity through two distinct effects: the reduction of the long closed state and modal switching to the high Po mode.

scholarly journals Properties of Single Voltage-gated Proton Channels in Human Eosinophils Estimated by Noise Analysis and by Direct Measurement

2003 ◽  
Vol 121 (6) ◽  
pp. 615-628 ◽  
Author(s):  
Vladimir V. Cherny ◽  
Ricardo Murphy ◽  
Valerij Sokolov ◽  
Richard A. Levis ◽  
Thomas E. DeCoursey
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Power Spectral Analysis ◽  
Open Probability ◽  
Proton Channels ◽  
Voltage Gated ◽  
Unitary Conductance ◽  
Power Spectral ◽  
Current Events ◽  
Channel Currents

Voltage-gated proton channels were studied under voltage clamp in excised, inside-out patches of human eosinophils, at various pHi with pHo 7.5 or 6.5 pipette solutions. H+ current fluctuations were observed consistently when the membrane was depolarized to voltages that activated H+ current. At pHi ≤ 5.5 the variance increased nonmonotonically with depolarization to a maximum near the midpoint of the H+ conductance-voltage relationship, gH-V, and then decreased, supporting the idea that the noise is generated by H+ channel gating. Power spectral analysis indicated Lorentzian and 1/f components, both related to H+ currents. Unitary H+ current amplitude was estimated from stationary or quasi-stationary variance, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{{\sigma}}_{\mathrm{H}}^{\mathrm{2}}\) \end{document}. We analyze \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{{\sigma}}_{\mathrm{H}}^{\mathrm{2}}\) \end{document} data obtained at various voltages on a linearized plot that provides estimates of both unitary conductance and the number of channels in the patch, without requiring knowledge of open probability. The unitary conductance averaged 38 fS at pHi 6.5, and increased nearly fourfold to 140 fS at pHi 5.5, but was independent of pHo. In contrast, the macroscopic gH was only 1.8-fold larger at pHi 5.5 than at pHi 6.5. The maximum H+ channel open probability during large depolarizations was 0.75 at pHi 6.5 and 0.95 at pHi 5.5. Because the unitary conductance increases at lower pHi more than the macroscopic gH, the number of functional channels must decrease. Single H+ channel currents were too small to record directly at physiological pH, but at pHi ≤ 5.5 near Vthreshold (the voltage at which gH turns on), single channel–like current events were observed with amplitudes 7–16 fA.

scholarly journals Tail End of the S6 Segment

2002 ◽  
Vol 120 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Shinghua Ding ◽  
Richard Horn
Keyword(s):  
Potassium Channels ◽  
Energy Barrier ◽  
Single Channel ◽  
Noise Analysis ◽  
Selectivity Filter ◽  
Open Probability ◽  
Cytoplasmic Extension ◽  
Activation Gate ◽  
Single Channel Currents ◽  
Channel Currents

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (Po,max) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces Po,max, whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (γ). Mutations of residue Y485 have no effect on the Rb+/K+ selectivity, suggesting a local effect on γ rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb+ ions for K+ ions reduces the amplitude of single channel currents and makes γ insensitive to mutations of Y485. These results suggest that Rb+ ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6T residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine

2002 ◽  
Vol 205 (23) ◽  
pp. 3729-3737 ◽  
Author(s):  
Fatima Atia ◽  
Irina Mountian ◽  
Jeannine Simaels ◽  
Etienne Waelkens ◽  
Willy Van Driessche
Keyword(s):  
Liquid Chromatography ◽  
System Analysis ◽  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Gel Filtration ◽  
Maximal Effect ◽  
Na Transport ◽  
Open Probability ◽  
Stimulatory Action

SUMMARY Effects of the extract of Nigella arvensis (NA) seeds on transepithelial Na+ transport were studied in cultured A6 toad kidney cells by recording short-circuit current (Isc),transepithelial conductance (GT), transepithelial capacitance (CT) and fluctuation in Isc. Apical application of NA extract had merely a small stimulatory effect on Na+ transport, whereas basolateral administration markedly increased Isc, GT and CT. A maximal effect was obtained at 500 μl l-1 of lyophilized NA extract. The increase in CT suggests that the activation of Isc occurs through the insertion of transport sites in the apical membrane. In experiments performed in the absence of Na+transport [apical Na+ was replaced by N-methyl-D-glucamine(NMDG+)], basolateral NA extract did not affect Isc and GT, indicating that Cl- conductance was not influenced. Noise analysis of Isc using 6-chloro-3,5-diaminopyrazine-2-carboxamide(CDPC) showed that NA extract reduced single-channel current(iNa) and decreased channel open probability(Po) but evoked a threefold increase in channel density(NT), which confirms the insertion of Na+channels. The separation of the compounds in the crude extract of NAwas performed by fast protein liquid chromatography (FPLC) on a Superdex 200 gel-filtration column and by reverse-phase high-pressure liquid chromatography(RPHPLC) on an μRPC C2/C18 SC2.1/10 column connected to a SMART system. Analysis of the purified active fraction by mass spectrometry demonstrated the presence of adenosine as the single organic compound in the extract that had a stimulatory effect on Na+ transport. In a separate series of experiments, we confirmed that 1 μmol l-1 adenosine had similar effects on the parameters of Na+ transport as did the NAextract. The action of adenosine was further identified by experiments in which NA extract was added after adenosine. In these experiments, NA extract did not affect Isc, GT or CT. These results clearly demonstrate an essential role of adenosine in the stimulatory action of NA extract.

scholarly journals Hormonal regulation of ENaCs: insulin and aldosterone

1998 ◽  
Vol 274 (5) ◽  
pp. C1373-C1379 ◽  
Author(s):  
Bonnie L. Blazer-Yost ◽  
Xuehong Liu ◽  
Sandy I. Helman
Keyword(s):  
Hormonal Regulation ◽  
Single Channel ◽  
Noise Analysis ◽  
Open Probability ◽  
Transport Pathway ◽  
Channel Density ◽  
Unstimulated Control ◽  
A6 Epithelia ◽  
20 Nm

Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current ( i Na), channel open probability ( P o), and functional channel density ( N T) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10–30 min the density of the pool of apical membrane ENaCs ( N T) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of i Na(10–20%) and decreases of P o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of i Na, P o, and N T were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

scholarly journals Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia

1998 ◽  
Vol 274 (4) ◽  
pp. C947-C957 ◽  
Author(s):  
Sandy I. Helman ◽  
Xuehong Liu ◽  
Kieron Baldwin ◽  
Bonnie L. Blazer-Yost ◽  
Willem J. Els
Keyword(s):  
Single Channel ◽  
Short Circuit ◽  
Noise Analysis ◽  
Early Time ◽  
Fold Increase ◽  
Time Dependent ◽  
Open Probability ◽  
Modified Method ◽  
A6 Epithelia ◽  
Stimulation Of

To study and define the early time-dependent response (≤6 h) of blocker-sensitive epithelial Na+channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current ( i Na) channel open probability ( P o), and channel density ( N T) under transient conditions of transport as measured by macroscopic short-circuit currents ( I sc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 μA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. N T varied linearly over a 70-fold range of transport (0.5–35 μA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of P o occurred during control (10–20% over 2 h) and aldosterone experimental periods (10–30% over 6 h). When the P o of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, P o was observed to vary inversely with I sc, falling from ∼0.5 to ∼0.15 at the highest rates of Na+ transport or ∼25% per 3-fold increase of transport. Because decreases of P o from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane N T.

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