Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

1988 ◽  
Vol 103 (2) ◽  
pp. 191-204 ◽  
Author(s):  
James S. Stoddard ◽  
Luis Reuss
Keyword(s):  
Time Dependence ◽  
Apical Membrane ◽  
Membrane Conductance ◽  
Gallbladder Epithelium ◽  
Current Clamp

Ba2+, TEA+, and quinine effects on apical membrane K+ conductance and maxi K+ channels in gallbladder epithelium

1990 ◽  
Vol 259 (1) ◽  
pp. C56-C68 ◽  
Author(s):  
Y. Segal ◽  
L. Reuss
Keyword(s):  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
K Channel ◽  
Dependent Manner ◽  
Gallbladder Epithelium ◽  
Concentration Dependent Manner ◽  
K Channels ◽  
Voltage Dependent ◽  
Channel Currents

The apical membrane of Necturus gallbladder epithelium contains a voltage-activated K+ conductance [Ga(V)]. Large-conductance (maxi) K+ channels underlie Ga(V) and account for 17% of the membrane conductance (Ga) under control conditions. We examined the Ba2+, tetraethylammonium (TEA+), and quinine sensitivities of Ga and single maxi K+ channels. Mucosal Ba2+ addition decreased resting Ga in a concentration-dependent manner (65% block at 5 mM) and decreased Ga(V) in a concentration- and voltage-dependent manner. Mucosal TEA+ addition also decreased control Ga (60% reduction at 5 mM). TEA+ block of Ga(V) was more potent and less voltage dependent that Ba2+ block. Maxi K+ channels were blocked by external Ba2+ at millimolar levels and by external TEA+ at submillimolar levels. At 0.3 mM, quinine (mucosal addition) hyperpolarized the cell membranes by 6 mV and reduced the fractional apical membrane resistance by 50%, suggesting activation of an apical membrane K+ conductance. At 1 mM, quinine both activated and blocked K(+)-conductive pathways. Quinine blocked maxi K+ channel currents at submillimolar concentrations. We conclude that 1) Ba2+ and TEA+ block maxi K+ channels and other K+ channels underlying resting Ga; 2) parallels between the Ba2+ and TEA+ sensitivities of Ga(V) and maxi K+ channels support a role for these channels in Ga(V); and 3) quinine has multiple effects on K(+)-conductive pathways in gallbladder epithelium, which are only partially explained by block of apical membrane maxi K+ channels.

scholarly journals Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium.

1990 ◽  
Vol 95 (5) ◽  
pp. 791-818 ◽  
Author(s):  
Y Segal ◽  
L Reuss
Keyword(s):  
Patch Clamp ◽  
Apical Membrane ◽  
Membrane Conductance ◽  
Membrane Depolarization ◽  
Membrane Voltage ◽  
K Channel ◽  
Gallbladder Epithelium ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Necturus Gallbladder

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.

scholarly journals cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.

1993 ◽  
Vol 102 (2) ◽  
pp. 177-199 ◽  
Author(s):  
J Copello ◽  
T A Heming ◽  
Y Segal ◽  
L Reuss
Keyword(s):  
Chloride Channels ◽  
Apical Membrane ◽  
Membrane Conductance ◽  
Intracellular Camp ◽  
Channel Blockers ◽  
Gallbladder Epithelium ◽  
Open Probability ◽  
Necturus Gallbladder ◽  
Cytosolic Side ◽  
Cl Channels

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- > SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.

scholarly journals Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium.

1991 ◽  
Vol 97 (5) ◽  
pp. 949-971 ◽  
Author(s):  
C U Cotton ◽  
L Reuss
Keyword(s):  
Cell Membrane ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Active Agent ◽  
Membrane Conductance ◽  
Membrane Resistance ◽  
Initial Increase ◽  
Bathing Solution ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.

scholarly journals Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

1992 ◽  
Vol 99 (2) ◽  
pp. 241-262 ◽  
Author(s):  
G A Altenberg ◽  
J S Stoddard ◽  
L Reuss
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Gallbladder Epithelium ◽  
K Channels ◽  
Necturus Gallbladder ◽  
Electrophysiological Effects ◽  
Paracellular Diffusion ◽  
Cl Conductance

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.

Ion transport in goby intestine: cellular mechanism of urotensin II stimulation

1985 ◽  
Vol 249 (2) ◽  
pp. G284-G293
Author(s):  
C. A. Loretz ◽  
M. E. Howard ◽  
A. J. Siegel
Keyword(s):  
Apical Membrane ◽  
Apical Cell ◽  
Phosphodiesterase Inhibitor ◽  
Membrane Conductance ◽  
Urotensin Ii ◽  
Apical Cell Membrane ◽  
Absorptive Cells ◽  
Ion Substitution ◽  
Columnar Cells ◽  
Leaky Epithelium

The Na- and Cl-absorbing goby posterior intestinal epithelium is composed predominantly of mitochondria-rich, tall columnar cells. Glass intracellular microelectrode recording technique was applied to absorptive cells of this relatively leaky epithelium to measure apical cell membrane potential difference (psi mc) and apical membrane fractional resistance. As determined by ion-substitution studies, absorptive cells are characterized by a large, Ba2+-inhibitable apical K conductance, which is a major factor determining psi mc and smaller Cl and Na conductances. Inhibition of the apical Na-Cl-coupled influx directly by furosemide or indirectly by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine produced hyperpolarization of psi mc, consistent with the greater apical membrane conductance to Cl than Na. The urophysial neurosecretory peptide urotensin II, which stimulates Na-Cl-coupled absorption, markedly depolarized psi mc in posterior intestinal tissues from 5% seawater-adapted gobies. This response is consistent with a stimulatory effect of urotensin II at the apical membrane carrier rather than at the basolateral Na-K-ATPase. Urotensin II is without effect on psi mc in tissues from seawater-adapted fish and somatostatin, a natural analogue of urotensin II, is without effect on tissues from fish adapted to either salinity. This specificity parallels that determined using radiotracer fluxes.

EGF and PGE2 inhibit rabbit CCD Na+ transport by different mechanisms: PGE2 inhibits Na(+)-K+ pump

1993 ◽  
Vol 264 (4) ◽  
pp. F670-F677 ◽  
Author(s):  
D. H. Warden ◽  
J. B. Stokes
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Membrane Voltage ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Na Transport ◽  
Flux Measurements ◽  
Epidermal Growth

The rabbit cortical collecting duct absorbs Na+ by a transport system comprised of an apical membrane Na+ channel and a basolateral membrane Na(+)-K(+)-adenosinetriphosphatase. The rate of Na+ absorption across this epithelium is acutely inhibited by several hormones and autacoids including epidermal growth factor (EGF) and prostaglandin E2 (PGE2). We used electrophysiological analysis to determine which Na+ transport mechanism is primarily regulated in response to EGF and PGE2. We used concentrations of EGF and PGE2 that inhibited Na+ absorption to a comparable degree. We assessed the effects of these agents on Na+ transport primarily by the calculated equivalent current; the validity of this indicator was verified using simultaneous tracer flux measurements. EGF and PGE2 had different effects on the intracellular electrophysiological parameters. EGF (in the presence of a cyclooxygenase inhibitor) hyperpolarized the apical membrane voltage in a manner analogous to the Na(+)-channel blocker amiloride, reduced the transepithelial conductance, and increased the fractional resistance of the apical membrane. In comparison, PGE2 depolarized the apical membrane voltage in a manner analogous to the Na(+)-K+ pump inhibitor ouabain, and caused no significant changes in transepithelial conductance or apical membrane conductance. The finding that EGF hyperpolarized the apical membrane indicates that this agent attenuates Na+ absorption by reducing apical Na+ entry due to a decrease in the magnitude of the apical membrane Na+ conductance. In contrast, the electrophysiological changes produced by PGE2 indicate primary inhibition of the basolateral Na(+)-K+ pump following PGE2 treatment.

Protamine alters apical membrane K+ and Cl- permeability in gallbladder epithelium

1987 ◽  
Vol 253 (5) ◽  
pp. C662-C671 ◽  
Author(s):  
S. M. Poler ◽  
L. Reuss
Keyword(s):  
Time Course ◽  
Apical Membrane ◽  
Membrane Resistance ◽  
Gallbladder Epithelium ◽  
Negative Change ◽  
High K ◽  
Solution Bathing ◽  
Transepithelial Voltage ◽  
Mucosal Side ◽  
Cl Permeability

Protamine addition to the solution bathing the mucosal side of Necturus gallbladder epithelium (25-100 mg/l) caused depolarization of both cell membranes, a mucosa-negative change in transepithelial voltage, an increase in the apical membrane resistance (Ra) followed by a decrease, and a monotonic increase in transepithelial resistance (Rt). In protamine (25 mg/l), the change in apical membrane voltage elicited by elevating mucosal solution [K+] from 2.5 to 92.5 mM was reduced from 66 +/-2 to 38 +/- 5 mV (P less than 0.001). The K+-induced fall in Ra was also reduced in protamine. These effects could also be elicited by elevating mucosal solution [K+] simultaneously with the addition of protamine and by transient addition of protamine during exposure to the high K+ medium. The effect of protamine on the electrodiffusive Cl- permeability of the apical membrane (PCl) was studied both in control and forskolin-treated tissues. In the absence of forskolin, the hyperpolarization of Vmc produced by lowering mucosal [Cl-] to 10 mM was reversed to a small depolarization; in forskolin, the initial depolarization produced by lowering [Cl-] was significantly increased. Finally, exposure to protamine in the absence of forskolin produced an initial fall in intracellular Cl- activity. Our results indicate that protamine decreases apical membrane K+ permeability and increases apical membrane PCl. The time course of the effects of protamine suggests the possibility of an initial effect on surface potential, followed by secondary actions mediated by intracellular events.

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