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M E Krouse

Publications and source records attributed to M E Krouse.

At least 19 recordsLinked to original sources
PubMed1997

Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR.

Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl(-)-dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl- channels. Typically, CF disrupts adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion but spares Ca(2+)-mediated secretion. However, in CF airway glands, Ca(2+)-mediated secretion is also greatly reduced. To determine the basis of Ca(2+)-mediated Cl- secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+ concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current (Isc) and transepithelial conductance (Gte) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (delta) in Isc and Gte, whereas forskolin stimulated variable and smaller increases. delta Isc was decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid, and calixarene. Isc was measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl- conductance (GCl) that was decreased by DPAC but not by DIDS. Apical GCl could be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive GCl pathway in the apical membrane and display approximately 60% of maximum conductance at rest. Thus elevated [Ca2+]i increases K+ conductance to force Cl- through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation.

The common delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and delta F508 in vivo. Incubation of transient and stable delta F508 transfectants with 10% glycerol induced a significant accumulation of delta F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued delta F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) delta F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.

Cells, Cultured↗
PubMed1996

Swelling and Ca2+-activated anion conductances in C127 epithelial cells expressing WT and delta F508-CFTR.

CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, delta F508-CFTR, or vector. Cell lines were compared quantitatively using an 125I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, 125I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were time- and voltage independent. All three lines responded to hypotonic challenge with large 125I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages. Unexpectedly, basal 125I efflux was significantly smaller in the delta F508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the cell line expressing delta F508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally selected cell lines that are unrelated to CFTR expression.

Animals↗
PubMed1996

Doxorubicin selection for MDR1/P-glycoprotein reduces swelling-activated K+ and Cl- currents in MES-SA cells.

To test the hypothesis that P-glycoprotein enhances swelling currents through regulation of volume-sensitive Cl- channels [recently termed VSOAC (volume-sensitive osmolyte and anion channel)], a human uterine sarcoma cell line (MES-SA) and its doxorubicin-selected counterpart (Dx5) were studied. P-glycoprotein mRNA and protein levels were detected only in Dx5 cells. However, whole cell patch-clamp experiments showed that swollen Dx5 cells (n = 5) produced smaller VSOAC currents than MES-SA cells (n = 4; 106 +/- 26 pA/pF vs. 232 +/- 76 pA/pF at 90 mV). In radioisotopic efflux experiments, both swelling-activated 125I (Cl-) currents (n = 15) and 86Rb (K+) currents (n = 8) were found to be two-to fourfold smaller in the Dx5 (high P-glycoprotein) cells. Inhibitors of P-glycoprotein showed no specificity for the doxorubicin-selected cells (Dx5). Dideoxyforskolin (100 microM) blocked swelling-activated 125I efflux equally in both cell lines, whereas 100 microM verapamil had no effect. Thus, in this cell line, selection for P-glycoprotein expression is associated with reduced swelling currents. These findings suggest that P-glycoprotein expression does not directly facilitate VSOAC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Delta F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines.

Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Dissociation of depolarization-activated and swelling-activated Cl- channels.

In many cells, patch excision and depolarization induce outwardly rectifying Cl- channels (ORDIC channels) whose function and normal mode of regulation are unknown. One possible function is the mediation of swelling-activated Cl- conductance, because in many cells rectifying Cl- currents are activated by cell swelling. However, swelling-activated Cl- channels in some epithelia have larger conductances than ORDIC channels and inactivate more rapidly, although both have similar anion selectivity and are blocked by stilbenes. Thus it has not been possible to determine whether the two types of channel current arise from distinct proteins or alternate states of a single protein. We studied 14 cell lines and found 2 lines, C127 mouse mammary epithelial cells and IEC-6 rat intestinal crypt cells, with very low levels of ORDIC channels. However, despite the near absence of ORDIC channels in these rodent cells, a large swelling-activated Cl-conductance was demonstrated by whole cell, efflux, and single-channel methods. Thus it is likely that ORDIC and swelling-activated channel currents arise from different channel proteins.

Animals↗
PubMed1994

Selection for MDR1/P-glycoprotein enhances swelling-activated K+ and Cl- currents in NIH/3T3 cells.

The relationship between multidrug resistance (MDR) P-glycoprotein expression and swelling-activated Cl- and K+ conductance was investigated in mouse NIH/3T3 fibroblasts and their colchicine-selected counterparts (COL1000, high P-glycoprotein). Whole cell patch-clamp and isotopic flux experiments confirmed that swelling-activated Cl- currents were induced by 20-30% bath dilution only in the MDR-expressing cell line. However, at bath dilutions > 30%, both cell lines developed Cl- currents that reached similar large magnitudes at higher dilution levels. Thus the apparent absolute difference in cell lines at lower dilutions is due to a shift in the response curve relating hypotonicity to Cl- conductance. At all dilutions and in both cell lines, the swelling-activated Cl- currents were outwardly rectifying, active at negative cell voltages, and inactivated at positive voltages. Verapamil (100 microM) and 1,9-dideoxyforskolin (100 microM), which inhibit P-glycoprotein drug transport, did not significantly inhibit the swelling-activated Cl- conductance efflux in the COL1000 cells also showed a leftward shift in the response curve to hypotonicity. These results indicate that response curve to hypotonicity. These results indicate that colchicine-selection for increased P-glycoprotein expression did not lead to the expression of swelling-activated Cl- channels, but instead enhanced a step in the pathway from bath dilution to regulatory volume decrease that is common to both K+ and Cl- channels.

3T3 Cells↗
PubMed1994

CFTR and other Cl- channels in human airway cells.

CFTR is the major Cl- channel in several epithelia, but its importance in airway cells and its interactions with other channels are still poorly understood. We studied the role of CFTR and other Cl- channels in two well-differentiated human airway cell lines--16HBE and Calu-3. Both lines form tight junctions, and the Calu-3 line exhibits large currents in the Ussing chamber that represent Cl- secretion. Apical membrane Cl- conductance in both cell lines in mediated primarily and possibly exclusively by CFTR, because (1) CFTR channels are almost the only anion channels observed in cell-attached patches from confluent cells, (2) whole-cell Cl- current has a linear, time-independent signature, and (3) blockers of many Cl- channels, such as DIDS, do not block Isc. Outwardly rectifying (ORDIC) channels were identified in both airway cell lines. Activation of ORDIC channels in Calu-3 cells required 3 conditions: (1) stimulation with isoproterenol, (2) excision and depolarization, and (3) prior treatment of the cell to disrupt tight junctions. To further assess the physiological significance of ORDIC channels, we compared cell lines which have > 10-fold difference in functional expression of ORDIC channels. ORDIC channel expression was not associated with swelling-activated Cl- currents.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Stilbenes stimulate T84 Cl- secretion by elevating Ca2+.

Basolateral but not apical application of 10-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to T84 monolayers produced a transient increase in short-circuit current (Isc), followed by a sustained inhibition. 4,4'-Dinitrostilbene-2,2'-disulfonic acid (DNDS) had no effect. The increase in Isc produced by DIDS represents Cl- secretion and appears to result from Ca2+ elevation, because in all respects except time course the response to DIDS mimicked the response to the Ca(2+)-elevating agent thapsigargin. Fura-2 measurements established that thapsigargin elevates Ca2+ in T84 cells, but Ca2+ responses to DIDS could not be established directly because DIDS absorbs strongly at the critical wavelengths. Responses to DIDS and thapsigargin were 1) blocked by bumetanide; 2) not blocked by basolateral Ba2+; 3) completely nonadditive; 4) strongly synergistic with basal levels of Isc or with Isc increases produced by elevating adenosine 3',5'-cyclic monophosphate (cAMP; with forskolin) or guanosine 3',5'-cyclic monophosphate (with heat-stable enterotoxin); and 5) reversibly abolished by removal of basolateral Ca2+. Interactions between Ca2+ and cAMP-elevating agents strongly support a model of Cl- secretion in which apical Cl- conductance is activated by cyclic nucleotides but not by Ca2+ while basolateral K+ channels are activated by Ca2+. In contrast with this mechanism, whole cell patch-clamp recordings of nonconfluent T84 cells indicated that DIDS and other Ca(2+)-elevating agents stimulated an increase in Cl- conductance. Thus increases in cytosolic free Ca2+ in nonconfluent T84 cells activate conductances that differ from those in confluent monolayers.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

CFTR channels in immortalized human airway cells.

The cystic fibrosis (CF) gene codes for CF transmembrane regulator (CFTR), a small-conductance linear Cl- channel, but numerous studies have identified a larger conductance, rectifying Cl- channel as the adenosine 3',5'-cyclic monophosphate (cAMP)-regulated channel that is defective in airway cells. We examined Cl- conductance in a bronchial epithelial cell line that expresses CFTR, 16HBE14o-, (CFTR+) and in an airway cell line that does not, 9HTEo-/S, (CFTR-). Ionomycin or hypotonic Ringer increased iodide efflux from both cell lines; however, forskolin increased iodide efflux or whole cell Cl- currents only in CFTR+ cells. Forskolin-stimulated whole cell currents were linear, voltage independent, and blocked by iodide. Cell-attached and outside-out patches from confluent CFTR+ but not CFTR- cells revealed 6-pS channels having linear current-voltage relations, permselectivity Cl > I (partial block by external iodide), and little or no inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate. The number of active channels per patch increased from 0.6 to 3.0 after forskolin. Channels closed after excision with tau = 4 s, but activity could be prolonged with ATP or protein kinase A plus ATP. Channels were modeled with one open and four closed states and show apparent cooperativity in gating. Rectifying Cl- channels previously implicated in CF were not seen in cell-attached recordings from either cell line but were abundant in excised patches from both cell lines. Thus CFTR channels are the pathway for cAMP-mediated Cl- conductance in these human airway cells, Ca2+ and swelling-induced channels do not require CFTR, and CFTR-cells display a CF phenotype.

Anions↗
PubMed1991

Cystic fibrosis gene expression is not correlated with rectifying Cl- channels.

Cystic fibrosis (CF) involves a profound reduction of Cl- permeability in several exocrine tissues. A distinctive, outwardly rectifying, depolarization-induced Cl- channel (ORDIC channel) has been proposed to account for the Cl- conductance that is defective in CF. The recently identified CF gene is predicted to code for a 1480-amino acid integral membrane protein termed the CF transmembrane conductance regulator (CFTR). The CFTR shares sequence similarity with a superfamily of ATP-binding membrane transport proteins such as P-glycoprotein and STE6, but it also has features consistent with an ion channel function. It has been proposed that the CFTR might be an ORDIC channel. To determine if CFTR and ORDIC channel expression are correlated, we surveyed various cell lines for natural variation in CFTR and ORDIC channel expression. In four human epithelial cell lines (T84, CaCo2, PANC-1, and 9HTEo-/S) that encompass the full observed range of CFTR mRNA levels and ORDIC channel density we found no correlation.

Base Sequence↗
PubMed1991

Cystic fibrosis, the CFTR, and rectifying Cl- channels.

The human genetic disease cystic fibrosis is caused by a single defective gene on chromosome 7 that codes for a 1480 amino acid protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The defect causes a profound reduction of Cl- permeability in several tissues, which in turn impairs salt absorption and fluid secretion. A 25-80 pS, rectifying Cl- channel has been targeted as the exclusive or primary channel affected in CF. However, we have found no evidence for significant activation or spontaneous activity of this channel in cell-attached patches of normal lymphoblasts or dog tracheal cells. However, in dog tracheal cells, we find lower conductance, linear Cl- channels that are spontaneously active in unstimulated cells and may show increased activity in stimulated cells. Attempts to correlate the expression of mRNA for the CFTR protein in various types of cells with the presence of the rectifying Cl- channel show a lack of correlation: i.e., depolarization-activated rectifying Cl- channesl have been found in excised, inside-out patches from all cell types that we have examined to date, but the CFTR mRNA has so far only been detected in a subset of epithelial cells.

Animals↗
PubMed1991

Peptide cotransmitter potentiates calcium channel activity in crayfish skeletal muscle.

The activity of 2 types of Ca2+ channels (38 and 14 pS in 137 mM Ba2+) in the plasma membrane of the crayfish tonic flexor muscle is modulated by the peptide proctolin. This peptide serves as a cotransmitter in 3 of the 5 excitatory tonic flexor motoneurons and greatly enhances tension after depolarization by the conventional neurotransmitter. Proctolin alone has no effect on these channels, but renders them capable of sustained activity following depolarization. After depolarization induces activity, 5 x 10(-9) M proctolin increases the open probability of the larger channel up to 50-fold due to a marked decrease in the mean channel closed time. There is also at least a 4-fold increase in the percentage of patches with active channels for the large channel and a 2-fold increase for the small channel. Proctolin modulation appears to occur via an intracellular messenger, possibly cAMP. The peptide's effect on channel activity is dose dependent in a manner that parallels its effect on tension. These results indicate that the activation of these channels and the resulting influx of Ca2+ into the muscle fiber play a role in the potentiation of tension in this muscle.

Animals↗
PubMed1989

Patch-clamp study of cultured human sweat duct cells: amiloride-blockable Na+ channel.

The reabsorptive duct of the eccrine sweat gland has a large transepithelial conductance consisting mainly of a high conductance to Cl- and a smaller, amiloride-blockable Na+ conductance (Bijman and Frömter 1986; Quinton 1986). Cells have been cultured from sweat ducts and their properties previously studied in Ussing chambers (Pedersen 1988) and with microelectrodes (Jones et al. 1988). We have now studied the ion channels present in excised, inside-out patches of human cultured sweat duct cells, and find a marked predominance of linear, 15 pS, amiloride-blockable, low selectivity, Na+ channels. Such channels were seen in 54/92 (59%) of the patches, with up to 7 channels recorded in a single patch. Other channel types were seen at much lower densities. The prevalence of an amiloride-blockable Na+ channel in cultured duct cells clearly distinguishes these cells from cultured sweat gland secretory cells, which lack such a channel.

Amiloride↗
PubMed1989

Ion channels in normal human and cystic fibrosis sweat gland cells.

Single-channel patch-clamp techniques were used to study the population of apical membrane ion channels in cultured sweat gland secretory cells from normal and cystic fibrosis subjects. Four types of anion channels and two types of cation channels were found. At physiological voltages, anion channels had chord conductances of 10, 18, 24, and greater than 200 pS. All had linear current-voltage relations except the 24 pS channel, which showed outward rectification. Cation channels had chord conductances of 5 and 18 pS, were linear, and were nonselective for a variety of cations. Channel types and proportions were equivalent in control, cystic fibrosis, and cystic fibrosis heterozygote cells. Beyond showing that the distribution of channel types remains unchanged in cystic fibrosis cells, the data provide a basis for comparison with cells cultured under different conditions, with other cell types, and with native tissues.

Adolescent↗
PubMed1985

Rates and equilibria for a photoisomerizable antagonist at the acetylcholine receptor of Electrophorus electroplaques.

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[alpha-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 microM for the cis and trans isomers, respectively. Flash-induced trans----cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately 10(8) M-1 s-1 and 60 s-1 at 20 degrees C; the Q10 is 3. Flash-induced cis----trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors.

Animals↗
PubMed1980

A covalently bound photoisomerizable agonist: comparison with reversibly bound agonists at Electrophorus electroplaques.

After disulphide bonds are reduced with dithiothreitol, trans-3- (alpha-bromomethyl)-3'-[alpha- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this "tethered agonist" shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 muM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to "open-channel blockade" bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3',bis-[alpha-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel's activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.

Animals↗