Artifactual expression of maxi-K+ channels in basolateral membrane of gallbladder epithelial cells

1993 ◽  
Vol 264 (5) ◽  
pp. C1128-C1136 ◽  
Author(s):  
J. Copello ◽  
F. Wehner ◽  
L. Reuss
Keyword(s):  
Epithelial Cells ◽  
Cell Membrane ◽  
Patch Clamp ◽  
Background Noise ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Gallbladder Epithelium ◽  
K Channels ◽  
Necturus Gallbladder ◽  
Apical Side

To patch clamp the basolateral cell membrane, sheets of Necturus gallbladder epithelium were stripped of the subepithelial tissue layers and affixed apical side down on cover slips coated with Cell-Tak [F. Wehner, L. Garretson, K. Dawson, Y. Segal, and L. Reuss. Am. J. Physiol. 258 (Cell Physiol. 27): C1159-C1164, 1990]. In 90% of the patches we observed K+ channels identical to the maxi-K+ channels previously demonstrated in the apical membrane (Y. Segal and L. Reuss. J. Gen. Physiol. 95: 791-818, 1990). To ascertain whether these channels were present in the native tissue, we carried out intracellular-microelectrode studies. We tested for activation of basolateral membrane K+ conductance by depolarization or by elevation of intracellular Ca2+ and for tetraethylammonium sensitivity of the basolateral membrane voltage and fractional resistance. The results were negative, indicating that maxi-K+ channels are not expressed in the basolateral membrane of the "intact" epithelium. Using the same intracellular-microelectrode protocol on the apical membrane, we demonstrated the presence of an apical K+ conductance attributable to maxi-K+ channels. Additional experiments revealed a Ba(2+)-sensitive basolateral K+ conductance in the native epithelium. We conclude that in the stripped preparation there is artifactual expression of maxi-K+ channels. In addition, the native basolateral membrane K+ channels either are not expressed in this preparation or have a low conductance and cannot be discerned from the background noise.

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.

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 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 Intracellular K+ activity and its relation to basolateral membrane ion transport in Necturus gallbladder epithelium.

1980 ◽  
Vol 76 (1) ◽  
pp. 33-52 ◽  
Author(s):  
L Reuss ◽  
S A Weinman ◽  
T P Grady
Keyword(s):  
Cell Membrane ◽  
Amphotericin B ◽  
Basolateral Membrane ◽  
Membrane Potentials ◽  
Equilibrium Potential ◽  
Gallbladder Epithelium ◽  
Bathing Medium ◽  
Necturus Gallbladder ◽  
Intracellular K Activity ◽  
K Activity

A study of the mechanisms of the effects of amphotericin B and ouabain on cell membrane and transepithelial potentials and intracellular K activity (alpha Ki) of Necturus gallbladder epithelium was undertaken with conventional and K-selective intracellular microelectrode techniques. Amphotericin B produced a mucosa-negative change of transepithelial potential (Vms) and depolarization of both apical and basolateral membranes. Rapid fall of alpha Ki was also observed, with the consequent reduction of the K equilibrium potential (EK) across both the apical and the basolateral membrane. It was also shown that, unless the mucosal bathing medium is rapidly exchanged, K accumulates in the unstirred fluid layers near the luminal membrane generating a paracellular K diffusion potential, which contributes to the Vms change. Exposure to ouabain resulted in a slow decrease of alpha Ki and slow depolarization of both cell membranes. Cell membrane potentials and alpha Ki could be partially restored by a brief (3-4 min) mucosal substitution of K for Na. Under all experimental conditions (control, amphotericin B, and ouabain), EK at the basolateral membrane was larger than the basolateral membrane equivalent emf (Eb). Therefore, the K chemical potential difference appears to account for Eb and the magnitude of the cell membrane potentials, without the need to postulate an electrogenic Na pump. Comparison of the rate of Na transport across the tissue with the electrodiffusional K flux across the basolateral membrane indicates that maintenance of a steady-state alpha Ki cannot be explained by a simple Na,K pump-K leak model. It is suggested that either a NaCl pump operates in parallel with the Na,K pump, or that a KCl downhill neutral extrusion mechanism exists in addition to the electrodiffusional K pathway.

Regulation of apical membrane ion transport in Necturus gallbladder

1992 ◽  
Vol 263 (1) ◽  
pp. C187-C193 ◽  
Author(s):  
J. L. Garvin ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Cell Swelling ◽  
Gallbladder Epithelium ◽  
Necturus Gallbladder ◽  
Camp Levels ◽  
Nacl Cotransport ◽  
Cl Conductance ◽  
Membrane Potential Difference ◽  
Membrane Ion Transport

Na and Cl movement through the apical membrane of Necturus gallbladder epithelium was investigated using electrophysiological and light microscopic measurements. Changes in membrane potential difference, fractional resistance of the apical membrane, and transepithelial resistance caused by changes in apical bath Cl concentration revealed the presence of a Cl conductance in the apical membrane of control tissues that was apparently not present in the preparations studied by other investigators. This Cl conductance was blocked by bumetanide (10(-5) M) or by the inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP) action, the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 0.5 mM). Treatment of the tissues with Rp-cAMPS also eliminated bumetanide-sensitive cell swelling in the presence of ouabain and activated an amiloride-sensitive swelling, changes consistent with inhibition of NaCl cotransport and the activation of Na-H and Cl-HCO3 exchange. We conclude that the mode of NaCl entry into Necturus gallbladder epithelial cells is determined by the level of cAMP. When cAMP levels are high, entry occurs by NaCl cotransport; when cAMP levels are low, parallel exchange of Na-H and Cl-HCO3 predominates. These observations explain the previous disagreements about the mode of NaCl entry into Necturus gallbladder epithelial cells.

Polarized expression of cAMP-activated chloride channels in isolated epithelial cells

1996 ◽  
Vol 271 (5) ◽  
pp. C1574-C1582 ◽  
Author(s):  
R. J. Torres ◽  
G. A. Altenberg ◽  
J. A. Cohn ◽  
L. Reuss
Keyword(s):  
Epithelial Cells ◽  
Patch Clamp ◽  
Fluorescent Dyes ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Freeze Fracture ◽  
Membrane Properties ◽  
Membrane Domains ◽  
Isolated Cells ◽  
Cl Conductance

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.

scholarly journals Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1274-1283 ◽  
Author(s):  
Christian Sänger ◽  
Elke Mühlberger ◽  
Elena Ryabchikova ◽  
Larissa Kolesnikova ◽  
Hans-Dieter Klenk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Surface Protein ◽  
Hemorrhagic Fever ◽  
Marburg Virus ◽  
Virus Surface ◽  
Mdckii Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

Bovine pancreatic duct cells express cAMP- and Ca(2+)-activated apical membrane Cl- conductances

1997 ◽  
Vol 273 (1) ◽  
pp. G204-G216 ◽  
Author(s):  
L. al-Nakkash ◽  
C. U. Cotton
Keyword(s):  
Epithelial Cells ◽  
Cell Membrane ◽  
Pancreatic Duct ◽  
Apical Membrane ◽  
Apical Cell ◽  
Primary Cultures ◽  
Apical Cell Membrane ◽  
Anion Secretion ◽  
Duct Epithelium ◽  
Cl Permeability

Secretion of salt and water by the epithelial cells that line pancreatic ducts depends on activation of apical membrane Cl- conductance. In the present study, we characterized two types of Cl- conductances present in the apical cell membrane of bovine pancreatic duct epithelial cells. Primary cultures of bovine main pancreatic duct epithelium and an immortalized cell line (BPD1) derived from primary cultures were used. Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ in intact monolayers of duct epithelium induced sustained anion secretion. Agonist-induced changes in plasma membrane Cl- permeability were accessed by 36 Cl- efflux, whole cell current recording, and measurements of transepithelial Cl- current across permeabilized epithelial monolayers. Elevation of intracellular cAMP elicited a sustained increase in Cl- permeability, whereas elevation of intracellular Ca2+ induced only a transient increase in Cl- permeability. Ca(2+)- but not cAMP-induced increases in Cl- permeability were abolished by preincubation of cells with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). N-phenylanthranilic acid (DPC; 1 mM) and glibenclamide (100 microM), but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 500 microM), inhibited the cAMP-induced increase in Cl- permeability. In contrast, DPC and DIDS, but not glibenclamide, inhibited the Ca(2+)-induced increase in Cl- permeability. We conclude from these experiments that bovine pancreatic duct epithelial cells express at least two types of Cl- channels, cAMP and Ca2+ activated, in the apical cell membrane. Because the Ca(2+)-activated increase in Cl- permeability is transient, the extent to which this pathway contributes to sustained anion secretion by the ductal epithelium remains to be determined.

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.

Coupled NaCl entry into Necturus gallbladder epithelial cells

1982 ◽  
Vol 243 (3) ◽  
pp. C140-C145 ◽  
Author(s):  
A. C. Ericson ◽  
K. R. Spring
Keyword(s):  
Epithelial Cells ◽  
Cell Volume ◽  
Apical Membrane ◽  
Ionic Composition ◽  
Basolateral Membrane ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Bathing Solution ◽  
Parallel Operation ◽  
Solute Content

NaCl entry into Necturus maculosus gallbladder epithelial cells was studied by determination of the rate of fluid movement into the cell when the Na+-K+-ATPase was inhibited by 10(-4) M ouabain in the serosal bathing solution. The cell swelling was due to continuing entrance of NaCl into the cell across the apical membrane, which increased the solute content of the cell; the resultant rise in cell osmolality induced water flow and cell swelling. The rate of swelling was 4.3% of the cell volume per minute, equivalent to a volume flow across the apical membrane of 1.44 x 10(-6) cm/s, similar in magnitude to the normal rate of fluid absorption by the gallbladder. We determined the mechanism of NaCl entry by varying the ionic composition of the mucosal bath; when most of the mucosal Na+ or Cl- was replaced, cell volume did not increase during pump inhibition. The rate of NaCl entry was a saturable function of Na+ or Cl- in the mucosal bathing solution with K1/2 values of 26.6 mM for Na+ and 19.5 mM for Cl-. The mode of NaCl entry was probably not the parallel operation of Na+-H+ and Cl(-)-HCO-3 exchangers because of the lack of effect of bicarbonate removal or of the inhibitors amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. NaCl entry was reversibly inhibited by bumetanide in the mucosal bathing solution. Transepithelial NaCl and water absorption is the result of the coupled, carrier-mediated movement of NaCl into the cell across the apical membrane and the active extrusion of Na+ by the Na+-K+-ATPase in the basolateral membrane.

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