Regulation of low-amiloride-affinity sodium channels in alveolar type II cells

1994 ◽  
Vol 267 (1) ◽  
pp. L94-L100 ◽  
Author(s):  
G. Yue ◽  
R. L. Shoemaker ◽  
S. Matalon
Keyword(s):  
Single Channel ◽  
Alveolar Type ◽  
Type Ii ◽  
Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Ionic Substitution ◽  
Beta Agonists ◽  
Single Channel Currents ◽  
Channel Currents

We determined the mechanisms by which beta-agonists increase sodium (Na+) currents across rat alveolar type II (ATII) cells grown in primary culture. When ATII cells were patched in the cell-attached mode using symmetrical Na+ solutions (150 mM Na(+)-glutamate), single-channel currents were observed for holding potentials between -80 and 30 mV (referenced to the pipette solution) with a single-channel conductance of 27 +/- 3 pS, a mean open time (tau 1) of 3.3 +/- 0.15 ms and an open probability (Po) of 0.36 +/- 0.06 (n = 7). Addition of 10 microM terbutaline into the bath increased tau 1 to 6.43 +/- 0.5 ms and Po to 0.62 +/- 0.06 (n = 7) without affecting channel conductance. Single-channel currents with a conductance of 25 +/- 2 pS were also recorded across ATII cells patched in the inside-out mode. Addition of 250 U/ml of protein kinase A (PKA), 1 mM ATP, and 5 mM MgCl2 in the bath solution (150 mM Na(+)-glutamate) increased the single channel tau 1 from 3.26 +/- 0.15 to 7.38 +/- 0.38 and Po from 0.41 +/- 0.06 to 0.72 +/- 0.07 (n = 6) without altering conductance. Addition of 1 microM amiloride or ethylisopropylamiloride (EIPA) in the pipette solution (150 mM Na(+)-glutamate) blocked single-channel activity almost completely. Ionic substitution experiments showed the relative permeability of Na+ to K+ and Na+ to Cl- to be 7:1 and 8:1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels

1998 ◽  
Vol 274 (4) ◽  
pp. L475-L484 ◽  
Author(s):  
Lucky Jain ◽  
Xi-Juan Chen ◽  
Lou Ann Brown ◽  
Douglas C. Eaton
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Cation Channel ◽  
Alveolar Type ◽  
Apical Surface ◽  
Type Ii ◽  
Patch Clamp Technique ◽  
Open Probability

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( P o), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the P o by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; P o, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; −42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( P o, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.

Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells

1993 ◽  
Vol 264 (3) ◽  
pp. C745-C754 ◽  
Author(s):  
A. Rich ◽  
J. L. Kenyon ◽  
J. R. Hume ◽  
K. Overturf ◽  
B. Horowitz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channels ◽  
Smooth Muscle Cells ◽  
Calcium Current ◽  
Single Channel ◽  
Muscle Cells ◽  
Open Probability ◽  
Boltzmann Function ◽  
Single Channel Currents ◽  
Channel Currents

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.

A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating

1997 ◽  
Vol 273 (4) ◽  
pp. F516-F529 ◽  
Author(s):  
Han Choe ◽  
Hao Zhou ◽  
Lawrence G. Palmer ◽  
Henry Sackin
Keyword(s):  
Single Channel ◽  
Ph Sensitivity ◽  
Channel Noise ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Hydrophobic Region ◽  
K Secretion ◽  
Single Channel Currents

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.

scholarly journals Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

2001 ◽  
Vol 118 (4) ◽  
pp. 341-353 ◽  
Author(s):  
Peter Proks ◽  
Charlotte E. Capener ◽  
Phillippa Jones ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Open Probability ◽  
Closed Intervals ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: Chloride imaging and cell-attached patch-clamp recordings

2000 ◽  
Vol 17 (2) ◽  
pp. 197-206 ◽  
Author(s):  
WALLACE B. THORESON ◽  
RON NITZAN ◽  
ROBERT F. MILLER
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Pipette Solution ◽  
Open Time ◽  
The Mean ◽  
Channel Properties ◽  
Distribution Fit

The present study uses cell-attached patch-recording techniques to study the single-channel properties of Ca2+ channels in isolated salamander photoreceptors and investigate their sensitivity to reductions in intracellular Cl−. The results show that photoreceptor Ca2+ channels possess properties similar to L-type Ca2+ channels in other preparations, including (1) enhancement of openings by the dihydropyridine agonist, (−)BayK8644; (2) suppression by a dihydropyridine antagonist, nisoldipine; (3) single-channel conductance of 22 pS with 82 mM Ba2+ as the charge carrier; (4) mean open probability of 0.1; (5) open-time distribution fit with a single exponential (τ0 = 1.1 ms) consistent with a single open state; and (6) closed time distribution fit with two exponentials (τc1 = 0.7 ms, τc2 = 25.4 ms) consistent with at least two closed states. Using a Cl−-sensitive dye to measure intracellular [Cl−], it was found that perfusion with gluconate-containing, low Cl− medium depleted intracellular [Cl−]. It was therefore possible to reduce intracellular [Cl−] by perfusion with a low Cl− solution while maintaining the extracellular channel surface in high Cl− pipette solution. Under these conditions, the single-channel conductance was unchanged, but the mean open probability fell to 0.03. This reduction can account for the 66% reduction in whole-cell Ca2+ currents produced by perfusion with low Cl− solutions. Examination of the open and closed time distributions suggests that the reduction in open probability arises from increases in closed-state dwell times. Changes in intracellular [Cl−] may thus modulate photoreceptor Ca2+ channels.

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.

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

1998 ◽  
Vol 275 (2) ◽  
pp. C535-C543 ◽  
Author(s):  
C. A. Obejero-Paz ◽  
M. Auslender ◽  
A. Scarpa
Keyword(s):  
Okadaic Acid ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell ◽  
A7r5 Cells ◽  
High Level ◽  
Single Channel Currents ◽  
Channel Currents

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

scholarly journals Effects of Ionic Strength on Gating and Permeation of TREK-2 K2P channels

2021 ◽  
Author(s):  
Linus J Conrad ◽  
Peter Proks ◽  
Stephen J Tucker
Keyword(s):  
Ionic Strength ◽  
Mechanism Of Action ◽  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Voltage Sensing ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Gating Process

In addition to the classical voltage-dependent behavior mediated by voltage-sensing-domains (VSD), a growing number of voltage-dependent gating behaviors are being described in ion channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel TREK2 responds to membrane voltage through a gating process that is mediated by the interaction of K+ with its selectivity filter. Recently, we have found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found that the channel is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is more complex.

Negative shift of cardiac Na+ channel kinetics in cell-attached patch recordings

1990 ◽  
Vol 258 (1) ◽  
pp. H247-H254 ◽  
Author(s):  
T. Kimitsuki ◽  
T. Mitsuiye ◽  
A. Noma
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Resting Potential ◽  
Na Channel ◽  
Channel Kinetics ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Pipette Solution ◽  
Single Channel Currents ◽  
Channel Currents

Na+ channel kinetics were studied by recording single-channel currents in the cell-attached patch configuration of the patch-clamp technique in single ventricular cells isolated from guinea pig hearts. The inactivation time course of ensemble currents was accelerated, and the peak amplitude increased temporarily and then decreased within a few minutes after the gigaohm seal formation. After reaching a new steady state, the inactivation-voltage relation was found to have shifted to more negative potentials. The potential of half-maximal inactivation was more negative by 20–31 mV from the resting potential or between -96 and -112 mV. The voltage dependency of the channel activation also shifted. Although the cell membrane was depolarized using the whole cell patch-clamp electrode and single-channel currents were recorded with an independent cell-attached electrode, the shift of the inactivation curve was also evident. Complete removal of Ca2+ using 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution failed to prevent the shift. Increasing Ca2+ to 10 mM, however, reduced magnitude of the shift significantly. Involvement of an increased membrane fluidity and surface potential of the glass pipette to the shift is discussed.

scholarly journals IKs ion-channel pore conductance can result from individual voltage sensor movements

2019 ◽  
Vol 116 (16) ◽  
pp. 7879-7888 ◽  
Author(s):  
Maartje Westhoff ◽  
Jodene Eldstrom ◽  
Christopher I. Murray ◽  
Emely Thompson ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Voltage Sensors ◽  
Repolarization Reserve ◽  
Accessory Subunits ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Open States ◽  
Channel Currents

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

Sign in / Sign up

Export Citation Format

Share Document