Mechanism of sodium hyperabsorption in cultured cystic fibrosis nasal epithelium: a patch-clamp study

1994 ◽  
Vol 266 (4) ◽  
pp. C1061-C1068 ◽  
Author(s):  
T. C. Chinet ◽  
J. M. Fullton ◽  
J. R. Yankaskas ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Patch Clamp ◽  
Single Channel ◽  
Apical Membrane ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Nasal Epithelial Cells ◽  
Na Permeability

Transepithelial Na+ absorption is increased two to three times in cystic fibrosis (CF) compared with normal (NL) airway epithelia. This increase has been associated with a higher Na+ permeability of the apical membrane of airway epithelial cells. Because Na+ absorption is electrogenic and abolished by amiloride, Na+ channels are thought to dominate the apical membrane Na+ permeability. Three Na+ channel-related mechanisms may explain the increase in apical Na+ permeability in CF cells: increased number of channels, increased single-channel conductance, and increased single-channel open probability. We compared the properties of Na(+)-permeable channels in the apical membrane of confluent preparations of human NL and CF nasal epithelial cells cultured on permeable supports. Na(+)-permeable channels were studied using the patch-clamp technique in the excised inside-out and cell-attached configurations. The same types of Na(+)-permeable channels were recorded in CF and NL cells. In excised patches, nonselective (Na+/K+) cation channels were recorded, and no differences between CF and NL were found in the properties, incidence, single-channel conductance, and single-channel open probability. In cell-attached patches, channels with a higher Na+ vs. K+ selectivity dominated. There was no difference between CF and NL cells in the incidence (18.8 vs. 21.4%, respectively) and conductance (17.2 +/- 2.8 vs. 21.4 +/- 1.5 pS, respectively) of Na(+)-permeable channels. However, the open probability was higher in CF cells compared with NL cells (30.0 +/- 3.4%, n = 6, vs. 15.0 +/- 3.9%, n = 13; P < 0.05). We conclude that, in CF nasal epithelial cells, the increase in Na+ permeability of the apical membrane results from an increase in the open probability of Na(+)-permeable channels in the apical membrane.

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.

Epithelial Ca2+ channels sensitive to dihydropyridines and activated by hyperpolarizing voltages

1994 ◽  
Vol 267 (1) ◽  
pp. C157-C165 ◽  
Author(s):  
H. Matsunaga ◽  
B. A. Stanton ◽  
F. A. Gesek ◽  
P. A. Friedman
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Apical Membrane ◽  
Channel Activity ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Nephron Segments ◽  
Convoluted Tubules ◽  
Ca2 Channels ◽  
In Cells

Parathyroid hormone (PTH) increases transcellular Ca2+ absorption in renal cortical thick ascending limbs and distal convoluted tubules (DCT). In cells isolated from these nephron segments, PTH increases Ca2+ uptake by a pathway that is sensitive to dihydropyridine-type agonists and antagonists (B. J. Bacskai and P. A. Friedman. Nature Lond. 347: 388-391, 1990). Patch-clamp techniques were used to identify Ca(2+)-permeable channels in DCT cells. Channel activity was detectable in cell-attached patches only in cells pretreated with PTH. Ca2+ channels exhibited prolonged open times (seconds), had a low single-channel conductance (2.1 pS), and open channel probability increased at hyperpolarizing voltages (-50 to -90 mV). Channel activity was sensitive to dihydropyridine-type compounds, nifedipine, and BAY K8644, as was Ca2+ uptake. However, Ca2+ entry was insensitive to verapamil or omega-conotoxin. These results demonstrate that these channels mediate PTH-stimulated apical membrane Ca2+ entry in DCT cells, which are the principal Ca(2+)-transporting cells of the kidney.

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.

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.

scholarly journals Anion permeation in an apical membrane chloride channel of a secretory epithelial cell.

1992 ◽  
Vol 99 (3) ◽  
pp. 339-366 ◽  
Author(s):  
D R Halm ◽  
R A Frizzell
Keyword(s):  
Epithelial Cell ◽  
Single Channel ◽  
Apical Membrane ◽  
Epithelial Cell Line ◽  
Low Permeability ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Conduction Pathway ◽  
Single Channel Currents ◽  
Secretory Epithelial Cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.

scholarly journals A Comparison of the Effect of Lead (Pb) on the Slow Vacuolar (SV) and Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2021 ◽  
Vol 22 (23) ◽  
pp. 12621
Author(s):  
Agnieszka Siemieniuk ◽  
Zbigniew Burdach ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Outward Currents ◽  
Inward Currents ◽  
Sv Channels ◽  
The One ◽  
The Impact

Little is known about the effect of lead on the activity of the vacuolar K+ channels. Here, the patch-clamp technique was used to compare the impact of lead (PbCl2) on the slow-activating (SV) and fast-activating (FV) vacuolar channels. It was revealed that, under symmetrical 100-mM K+, the macroscopic currents of the SV channels exhibited a typical slow activation and a strong outward rectification of the steady-state currents, while the macroscopic currents of the FV channels displayed instantaneous currents, which, at the positive potentials, were about three-fold greater compared to the one at the negative potentials. When PbCl2 was added to the bath solution at a final concentration of 100 µM, it decreased the macroscopic outward currents of both channels but did not change the inward currents. The single-channel recordings demonstrated that cytosolic lead causes this macroscopic effect by a decrease of the single-channel conductance and decreases the channel open probability. We propose that cytosolic lead reduces the current flowing through the SV and FV channels, which causes a decrease of the K+ fluxes from the cytosol to the vacuole. This finding may, at least in part, explain the mechanism by which cytosolic Pb2+ reduces the growth of plant cells.

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.

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