Mechanism of the defect in gap-junctional communication by expression of a connexin 26 mutant associated with dominant deafness.

Gap-junctional channels (connexin oligomers) are large-diameter aqueous pores formed by head-to-head association of two gap-junctional hemichannels, one from each of the adjacent cells. Profound hearing loss of genetic origin is common, and mutations of connexin 26 (Cx26) are the most frequent cause of this disorder. The Cx26 R75W mutant has been associated with disruption of cell-to-cell communication and profound hearing loss, but the mechanism of the gap-junctional defect is unknown. Here, we show that Cx26 R75W forms gap-junctional hemichannels that display altered voltage dependency and reduced permeability, and which cannot form functional gap-junctional channels between neighboring cells. The R75W phenotype is dominant at the gap-junction channel but not at the hemichannel level. Therefore, the absence of gap-junctional communication caused by R75W expression is due to defective gap-junction formation by functional hemichannels.

P-glycoprotein-associated Cl- currents are activated by cell swelling but do not contribute to cell volume regulation.

P-glycoprotein-associated Cl- conductance is activated by cell swelling; the ensuing Cl- efflux is thought to contribute to cell volume regulation. We tested this hypothesis in human breast cancer cells transfected with human mdr1 complementary DNA, which display P-glycoprotein-associated, swelling-activated Cl- currents. The Cl- electrochemical driving force favors Cl- efflux, but there was no appreciable Cl- loss or regulatory volume decrease (both assessed with fluorescent dyes) during the exposure to hyposmotic solution. Calculations indicate that the swelling-activated Cl- current is insufficient to cause a significant Cl- efflux. Hence, regulatory volume decrease is not a function of P-glycoprotein.

Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration.

Intracellular microelectrode techniques and extracellular pH measurements were used to study the dependence of apical Na+/H+ exchange on mucosal and intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na(+)-depleted tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM increased aNai; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating [Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na+ entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a histidine-specific reagent, reduced mucosal acidification by 58 +/- 4 or 39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%, respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi, upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by titration of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results suggest that external Na+ and H+ interact with one site of the Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The external titratable group seems to be an imidazolium, which is apparently different from the amiloride-binding site. The dependence of Na+ entry on pHi supports the notion that the Na+/H+ exchanger is operational under normal transport conditions.

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

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.

PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR.

Mutations of the CFTR, a phosphorylation-regulated Cl(-) channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.

Cell swelling activates the K+ conductance and inhibits the Cl- conductance of the basolateral membrane of cells from a leaky epithelium.

Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral membrane conductances for Cl- (GClb) and K+ (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K+/Cl- selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at approximately 30 s, return to baseline within approximately 90 s), but chelation of cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10-12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering basolateral solution [Cl-], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases GKb and decreases GClb. These two effects appear to be linked, i.e., the increase in GKb produces membrane hyperpolarization, which in turn reduces GClb. (b) Hyperosmotic shrinkage has the opposite effects on GKb and GClb. (c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.

Inhibition of drug transport by genistein in multidrug-resistant cells expressing P-glycoprotein.

It has been claimed that the flavonoid genistein could be used to distinguish multidrug-resistant tumors expressing the multidrug resistance-associated protein (MRP) from those expressing P-glycoprotein (Pgp). Genistein would be block drug transport by MRP without affecting Pgp-mediated drug transport. However, we found that exposure to 200 microM genistein elicited an elevation in intracellular accumulation of rhodamine 123 (R123) and daunorubicin (DNR) in Pgp-expressing cell lines. Genistein inhibited R123 efflux in a rapidly reversible manner (ca. 2 min). The flavonoid also decreased photoaffinity labeling of Pgp by [3H]azidopine, a Pgp substrate. The present results show that genistein interacts with Pgp and inhibits Pgp-mediated drug transport. Hence, genistein cannot be used in simple assays to distinguish MRP- and Pgp-expressing cells.

Mechanism of inhibition of P-glycoprotein-mediated drug transport by protein kinase C blockers.

P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport hy mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.

Sequestration of doxorubicin in vesicles in a multidrug-resistant cell line (LZ-100).

A multidrug-resistant Chinese hamster cell line, LZ-8, was subcultured in increasing levels of doxorubicin (DOX) until capable of growth in 100 micrograms/mL DOX. This new derivative, designated LZ-100, is the most DOX-resistant line in the LZ series, based on a comparison of Ki-1 values from cell survival studies. This increased level of drug resistance in LZ-100 cells did not result from (i) higher levels of P-glycoprotein (P-gp) in the plasma membrane compared with LZ-8 cells, since this protein constitutes approximately 20% of the total plasma membrane protein in both cell lines, or (ii) more efficient drug pumping by the same amount of P-gp, since efflux of rhodamine 123 and DOX was comparable in the two cell lines. However, an altered drug distribution was observed in LZ-100 cells compared with wild-type V79 cells; in LZ-100 cells DOX was largely excluded from the nucleus and was sequestered in vesicles in the cytoplasm. The number of vesicles per cell seen after DOX exposure corresponded with the level of drug resistance achieved by the LZ cell lines studied. DOX concentration-response experiments revealed that vesicle formation exhibited a biphasic relationship, with an initial rapid increase followed by a plateau where no further increase was observed. Time-course studies in LZ-100 cells revealed that the maximum number of DOX-containing vesicles per cell occurred 3-4 hr following initiation of DOX treatment. Radiation exposure (10 Gy) immediately preceding DOX treatment decreased the number of vesicles formed in LZ-100 cells by more than one-half and altered the subcellular distribution of DOX from an almost exclusively cytoplasmic to a homogeneous nuclear/cytoplasmic distribution. This redistribution was not a result of radiation inhibition of P-gp efflux. The inhibitory effect of radiation on vesicle formation increased with increasing radiation dose up to 10 Gy. Drug-containing vesicles were also observed in LZ-100 cells following exposure to mitoxantrone or daunorubicin (to which LZ-100 cells are also resistant), but fewer vesicles were observed than with DOX. These studies demonstrate that the drug sequestration phenomenon (i) occurs in cells exhibiting widely different levels of drug resistance, (ii) correlates with the level of drug resistance in LZ cell lines, (iii) occurs rapidly following exposure to DOX, mitoxantrone, or daunorubicin, and (iv) can be inhibited by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular alkalosis induced by increasing extracellular potassium. Ionic dependence and effects of amiloride and DIDS.

The intracellular alkalinization produced when extracellular potassium concentration is increased above its normal levels was studied in the rat diaphragm muscle by determination of the steady-state distribution of [14C]-5,5-dimethyl-2,4-oxazolidinedione (DMO). Replacement of external Na+ with sucrose and Mg2+ or N-methyl-D-glucamine prevented the rise in intracellular pH. Amiloride (1 mM) also abolished the elevation of intracellular pH, while the removal of external Cl- (replaced by gluconate) or addition of 0.1 mM 4-acetamido-4'-diisothyocyanostilbene-2,2-disulfonic acid (DIDS) did not prevent intracellular alkalinization from taking place. These results suggest that in the rat diaphragm muscle a Na(+)-dependent, amiloride-sensitive transport mechanism, perhaps Na+/H+ exchange, plays a major role in the K(+)-induced intracellular alkalinization. This mechanism might account for the metabolic acidosis produced by hyperkalemia.

P-glycoprotein is not a swelling-activated Cl- channel; possible role as a Cl- channel regulator.

1. The whole-cell configuration of the patch-clamp technique was used to determine if P-glycoprotein (Pgp) is a swelling-activated Cl- channel. 2. Hamster pgp1 cDNA was transfected into a mouse fibroblast cell line resulting in expression of functional Pgp in the plasma membrane. This cell line was obtained without exposure to chemotherapeutic agents. 3. Swelling-activated whole-cell Cl- current (ICl,swell) was elicited by lowering the bath osmolality. ICl,swell was characterized in detail in the pgp1-transfected mouse cell line and compared with that of its parental cell line. Expression of Pgp did not modify the magnitude or properties of ICl,swell, except that addition of the anti-Pgp antibody C219 to the pipette solution inhibited this current by 75% only in the Pgp-expressing cells. 4. ICl,swell in the mouse Pgp-expressing cell line was compared with that in a Pgp-expressing hamster fibroblast cell line. The characteristics of ICl,swell (voltage dependence, blocker sensitivity, anion selectivity sequence, requirement for hydrolysable ATP) in Pgp-expressing cells were different between the two cell lines. These results suggest that the channel(s) responsible for ICl,swell are different between the two cell lines. In addition, C219 inhibited ICl,swell in both Pgp-expressing cell lines, even though they seem to express different swelling-activated Cl- channels. 5. We conclude that firstly, Pgp is not a swelling-activated Cl- channel; secondly, it possibly functions as a Cl- channel regulator; and thirdly, ICl,swell is underlined by different Cl- channels in different cells.

Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts.

The substitution of gluconate for Cl- is commonly used to characterize Cl- transport or Cl--dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl- on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl- (Cl--Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl- by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl--Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl-. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport.

Muscarinic stimulation of gallbladder epithelium. III. Antagonism of cAMP-mediated effects.

Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) levels in Necturus gallbladder (NGB) epithelium activates an apical membrane Cl- conductance and decreases transepithelial fluid transport (Jv). Acetylcholine (ACh), which had no effects on Jv by itself, antagonized the electrophysiological effects of forskolin (FSK) and theophylline and the decrease in Jv produced by FSK. By itself, ACh had no effects on basal cAMP levels but antagonized the increases in cAMP induced by FSK and theophylline. ACh had no effect on phosphodiesterase activity and prevented both the electrophysiological response and the elevation in cAMP by theophylline. In conclusion, the effect of ACh is mediated by inhibition of adenylate cyclase. A pertussis toxin (PTX)-sensitive G protein may mediate inhibition of adenylate cyclase because pretreatment with PTX prevented the reversal of the electrophysiological effects of FSK by ACh, and PTX catalyzed the ribosylation of cell membranes from NGB epithelium. ACh could have a physiological role in modulating the effects of secretagogues that act via elevation of cAMP levels.

Muscarinic stimulation of gallbladder epithelium. II. Fluid transport, cell volume, and ion permeabilities.

Activation of muscarinic receptors in the fluid-absorptive epithelium of the Necturus gallbladder elevates cytosolic Ca2+ concentration, transiently hyperpolarizes the cell membrane voltages, and decreases the apparent fractional resistance of the apical membrane [G. A. Altenberg, M. Subramanyam, J. S. Bergmann, K. M. Johnson, and L. Reuss. Am. J. Physiol. 265 (Cell Physiol. 34): C1604-C1612, 1993]. In these studies, we show that at the peak of the hyperpolarization both apical and basolateral membrane resistances (Ra and Rb, respectively) decreased, but in 2-3 min Ra returned to control values while Rb rose to a level approximately 60% higher than control. The acetylcholine (ACh)-induced decrease in Ra is caused by activation of apical membrane maxi K+ channels secondary to elevation of cytosolic Ca2+ concentration. The increase in Rb is due to decreases in K+ and Cl- conductances. ACh had no effects on cell KCl content or water volume, although K+ conductance transiently increased. These results can be explained by the changes in basolateral membrane conductances. ACh did not alter fluid absorption. In conclusion, ACh has complex time-dependent effects on K+ and Cl- electrodiffusive permeabilities without measurable changes in cell volume or in the rate of transepithelial fluid transport.

Mechanism of acetazolamide-induced rise in renal vascular resistance assessed in the dog whole kidney.

The reduction in renal blood flow (RBF) and glomerular filtration rate (GFR) observed after the administration of the carbonic anhydrase inhibitors acetazolamide and benzolamide had been explained as due to activation of the tubuloglomerular feedback mechanism. If correct, pharmacologic blockade of this pathway should prevent the development of renal vasoconstriction with the carbonic anhydrase inhibitors. Thus, the current study evaluates in the dog whole kidney the effect of acetazolamide (20 mg/kg body weight) in the presence or absence of furosemide (5 mg/kg body weight), a drug which blocks the tubuloglomerular feedback. Acetazolamide resulted in a large increase in urinary bicarbonate excretion accompanied by a significant reduction in GFR (16%) and RBF (18%). By contrast with the effects of acetazolamide, furosemide did not alter GFR and increased RBF. In addition, the loop diuretic induced a large chloruresis without changes in urinary bicarbonate excretion. The infusion of acetazolamide in furosemide-treated dogs resulted in a significant increment in renal bicarbonate excretion and in a significant reduction in the levels of both GFR (28%) and RBF (13%). Therefore, furosemide pretreatment did not block the effects of acetazolamide on renal hemodynamic parameters. Consequently, the acetazolamide-induced reduction in both GFR and RBF cannot be accounted for by changes in chloride levels in the juxtaglomerular region due to enhanced salt transport in the macula densa/distal nephron. The increased renal vascular resistance observed with acetazolamide might occur by either a direct effect of this agent on the renal circulation or as a result of changes in intrarenal pressure secondary to the inhibition of proximal fluid reabsorption.

Cell-volume changes and ion conductances in amphibian gallbladder epithelium.

Cell-volume regulation after hyposmotic swelling is common in transporting epithelial cells. Acute regulatory volume decrease is mediated in several cell types by loss of K(+) and Cl(-) via channels either activated by the swelling phenomenon or active in the cell membrane prior to swelling. In the last two decades, it has become clear that many features of cell-volume regulation mediated by ion fluxes vary considerably among cell types. In some instances, the ion channel activation, although demonstrable, is insufficient to account, on the basis of ion fluxes, for the measured cell volume changes. Here we review the case of a salt-transporting epithelium in which hyposmotic cell swelling activates plasma membrane K(+) channels, but at the same time inhibits Cl(-) channels, thus preventing an acute volume-regulatory response. The sequence of events following cell swelling appears to be as follows: K(+) channels are activated by membrane stretch, the cell membrane hyperpolarizes (the voltage moves closer to the K(+) equilibrium potential) and this hyperpolarization reduces the Cl(-) conductance, likely by a reduction in open probability of voltage-sensitive Cl(-) channels. The significance of this phenomenon may be related to the preservation of intracellular Cl(-) rather than cell volume, or to a physiological 'need' to prevent shrinkage of cells stimulated by hormones or other mediators.

P-glycoprotein-associated chloride currents revealed by specific block by an anti-P-glycoprotein antibody.

The relationships between P-glycoprotein (PGP) expression and plasma membrane ion currents activated by cell swelling were studied in several cell lines by use of the whole cell configuration of the patch-clamp technique. Swelling-activated Cl- currents (ICls) had similar characteristics independently of whether PGP was expressed. Addition of the anti-PGP monoclonal antibody C219 or its Fab fragment to the pipette solution prevented ICls in cells expressing functional PGP (assessed by immunoblots, immunofluorescence, and transport of rhodamine 123) but not in cells lacking PGP expression. A peptide analogue of the C219 epitope abolished the effect of C219. Other anti-PGP antibodies and mouse immunoglobulin G were ineffective. C219 did not alter swelling-activated cation currents. Inasmuch as ICls is present in cells that do not express PGP and C219 has no effect on ICls in these cells, we conclude that PGP is not required for the ICls phenotype. However, when expressed in the plasma membrane, PGP is involved, directly or indirectly, in ICls but not in swelling-activated K+ currents.

Preservation of structural and functional polarity in isolated epithelial cells.

We describe a method to isolate epithelial cells from gallbladders of Necturus maculosus with preserved structural and functional polarity. Isolation was carried out with a mixture of collagenase and protease, with only a brief exposure to a divalent-cation-free medium. About 40% of the isolated epithelial cells had a "figure-eight" shape and retained metabolic and cell membrane integrity. Figure-eight cells display features consistent with preserved polarity for several hours, including the following: 1) the "apical" and "basolateral" membrane domains were differentially labeled by a hydrophobic fluorescent dye; 2) freeze fracture electron microscopy verified two plasma membrane domains differing in the presence of microvilli and folds and separated by tight junctions; 3) proteins such as ZO-1, NHE3, and Na(+)-K(+)-ATPase remained localized in the junctional, apical, and basolateral regions, respectively; 4) after apical surface exposure to wheat germ agglutinin, the label remained in the apical membrane after cell isolation; and 5) patch-clamp experiments demonstrated polarized expression of K+ channels. Polarity was rapidly lost after removal of extracellular Ca2+, exposure to trypsin, or ATP depletion. Therefore, this preparation allows for structural and functional studies of epithelial transport in single cells retaining the essential features present in the assembled epithelium.

Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein.

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP-NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.

Reversal by indomethacin of renal effects of dopamine in subjects with normal renal function.

In five subjects with normal renal function, intravenous dopamine, at a rate of 6 ug/kg/min, produces a renal vasodilation, increase in Na excretion and reduction in urinary osmolality, without modification in glomerular filtration rate. These effects were reversed by intravenous indomethacin (2 mg/kg), suggesting that the renal effects of dopamine might depend on normal prostaglandins production.