Biomedical subjects

H R de Jonge

Publications and source records attributed to H R de Jonge.

At least 19 recordsLinked to original sources

PubMed2004

[Expression of the cystic fibrosis gene in the lungs].

Cystic fibrosis (CF) is one of the most common hereditary diseases with a potentially lethal outcome. CF is caused by mutations in a gene on chromosome 7 that encodes for a polypeptide called 'cystic fibrosis transmembrane conductance regulator' (CFTR). Pulmonary complications in CF are a direct consequence of a fault in the production or function of CFTR. The consequences of these faults are a change in the viscosity and volume of the air-surface liquid in the airways and possible malfunctioning of the immunological defence system. The result is stasis of mucus and obstruction of the smaller airways. Secondary to this process, a (chronic) infection with an uncontrolled inflammatory response, leads to destruction of lung tissue and a reduction in lung function. Mutations in CFTR can be classified in five types, which reflect the level of production or the function of CFTR. This classification permits discrimination between mild and severe mutations. The clinical image and the course of CF are often related to the combination of mutations present. This may explain, in part, why some CF patients are diagnosed at a later age with a normal sweat test and a clinical image that is predominantly limited to respiratory symptoms.

Chromosomes, Human, Pair 7↗

PubMed2001

Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings.

To investigate the impact of chloride (Cl(-)) permeability, mediated by residual activity of the cystic fibrosis transmembrane conductance regulator (CFTR) or by other Cl(-) channels, on the manifestations of cystic fibrosis (CF), we determined Cl(-) transport properties of the respiratory and intestinal tracts in Delta F508 homozygous twins and siblings. In the majority of patients, cAMP and/or Ca(2+)-regulated Cl(-) conductance was detected in the airways and intestine. Our finding of cAMP-mediated Cl(-) conductance suggests that, in vivo, at least some Delta F508 CFTR can reach the plasma membrane and affect Cl(-) permeability. In respiratory tissue, the expression of basal CFTR-mediated Cl(-) conductance, demonstrated by 30% of Delta F508 homozygotes, was identified as a positive predictor of milder CF disease. In intestinal tissue, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-insensitive (DIDS-insensitive) Cl(-) secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive Cl(-) secretion was observed mainly in more severely affected patients. The more concordant Cl(-) secretory patterns within monozygous twins compared with dizygous pairs imply that genes other than CFTR significantly influence the manifestation of the basic defect.

Adolescent↗

PubMed2000

Protein N-myristoylation: critical role in apoptosis and salt tolerance.

N-myristoylation is a covalent protein modification that can promote the association of proteins with membranes. De Jonge, Hogema, and Tilly discuss how N-myristoylation may be involved in triggering Fas ligand-induced apoptosis in mammals, and in adapting to conditions of high salt in plants. The pro-apoptotic protein BID is unique in that its proteolytic cleavage product, tBID, is posttranslationally myristoylated. In contrast, the plant accessory protein SOS3 undergoes "classical" cotranslational N-myristoylation. N-myristoylation is essential for the proper functioning of these proteins in regulating the signaling pathways (apoptosis and adaptation to salt stress, respectively) in which they are involved.

Animals↗

PubMed2000

Pitfalls in the immunohistochemical localization of the cystic fibrosis transmembrane conductance regulator in paraffin embedded sweat glands.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the hereditary disease cystic fibrosis. The most frequent mutant deltaF508 has been shown in vitro to be retained in the endoplasmic reticulum. Ex vivo studies using immunohistochemical labelling in cryofixed skin biopsies have confirmed the mislocalization of deltaF508 CFTR in sweat glands. The purpose of this study was to test CFTR antibodies in paraffin-embedded skin biopsies to take advantage of the superior tissue preservation as compared to cryofixation. A panel of 7 CFTR antibodies was applied to skin sections of healthy controls and of cystic fibrosis patients homozygous for the deltaF508 mutation. Sweat gland labelling consistent with CFTR localization and different between control and cystic fibrosis tissue was obtained with 2 antibodies. Conventional staining controls confirmed the labelling specificity. The antibodies were subsequently tested in a series of 237 sections of 16 biopsy specimens. However, the sweat gland labelling pattern proved not to be dependent on CFTR genotype. This finding was the sole indicator of non-specificity of the staining which was revealed only by the size of our random sample. Our results emphasize that CFTR immunolabelling following formalin fixation has to be interpreted with the utmost caution.

Antibodies, Monoclonal↗

PubMed2000

Regulated trafficking of the CFTR chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.

Animals↗

PubMed2000

Signalling mechanisms involved in volume regulation of intestinal epithelial cells.

Most mammalian cells have developed compensatory mechanisms to respond to the variable osmotic stress caused by changes in the concentrations of intracellular osmo-active substances (e.g. glucose, amino acids, lactate) or by variations in the osmolarity of the surrounding medium. In response to osmotic cell swelling, the Regulatory Volume Decrease (RVD) is triggered and directs a reduction in the tonicity of the cell by the concerted opening of cation and anion selective ion channels. To date, the K(+) and Cl(-) conductances activated upon hypo-osmotic stimulation have been characterised electrophysiologically in many different cell systems. The molecular identity of the channels however, as well as the mechanism(s) involved in their activation have not yet been fully clarified and may differ between cell types. In this review, we will evaluate the different signalling pathways activated by osmotic cell swelling and discuss their putative role(s) in ion channel regulation, in maintaining cellular volume homeostasis, and in auto- and paracrinic signal transduction, with emphasis on intestinal epithelial cells.

Animals↗

PubMed1999

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II.

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Ibeta or a membrane-bound cGK II/Ibeta chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Animals↗

PubMed1999

LLC-PK1 cells as a model system to study proximal tubule transport of water and other compounds relevant for renal stone disease.

LLC-PK1 cells were cultured on a permeable support in a two-compartment culture system. Confluent monolayers received an ultrafiltrate-like solution at the apical side and a plasma-like solution at the basolateral side. The distribution of various solutes, including phosphate, calcium, and oxalate over both compartments was measured in time. The transport of water was monitored by alterations in fluid concentrations of radiolabeled inulin. Bicarbonate, glucose, and phosphate were transported rapidly from the apical to basolateral side of the monolayer. Sodium and chloride were reabsorbed without major consequences for the osmolality in the apical and basal fluid. Calcium and potassium were also reabsorbed, but to a smaller extent than sodium. The luminal concentration of oxalate gradually increased to values that were at least three times higher (12.0+/-0.4 micromol/l) than those in the contraluminal fluid (3.8+/-0.1 micromol/l). However, since the luminal rise of oxalate completely matched the rise of inulin in the apical fluid this appeared to be the passive consequence of active water reabsorption rather than of net directed oxalate transport. The LLC-PK1 model could prove useful to study the regulation of proximal tubule water transport and its effect on luminal stone salt concentrations under different physiological conditions.

Animals↗

PubMed1998

Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in intestine 407 cells involves the Ras/Raf-signalling pathway.

Human Intestine 407 cells respond to hypo-osmotic stress with a rapid stimulation of compensatory ionic conductances accompanied by a transient increase in the activity of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2. In this study, we examined the upstream regulators of hypotonicity-induced Erk-1/Erk-2 activation and their possible role in cell-volume regulation. The hypotonicity-provoked Erk-1/Erk-2 activation was greatly reduced in cells pretreated with the specific mitogen-activated/Erk-activating kinase inhibitor PD098059 and was preceded by a transient stimulation of Raf-1. Pretreatment of the cells with PMA, GF109203X, wortmannin or Clostridium botulinum C3 exoenzyme did not appreciably affect the hypotonicity-provoked Erk-1/Erk-2 stimulation, suggesting the osmosensitive signalling pathway to be largely independent of protein kinase C and p21(rho). In contrast, expression of dominant negative RasN17 completely abolished the hypotonicity-induced Erk-1/Erk-2 activation. Stimulation of the swelling-induced ion efflux was independent of activation of these mitogen-activated protein kinases, as revealed by hypotonicity-provoked isotope efflux from 125I-- and 86Rb+-loaded cells after pretreatment with PD098059 and after expression of RasN17. In addition, the epidermal-growth-factor-induced potentiation of the hypotonicity-provoked anionic response did not depend on the increase in Erk-1/Erk-2 activity but, instead, was found to depend on Ca2+ influx. Taken together, these results indicate that hypotonic stress induces Erk-1/Erk-2 activation through the Ras/Raf-signalling pathway, and argue against a direct role for this pathway in cell-volume control.

ADP Ribose Transferases↗

PubMed1998

Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation.

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.

Animals↗

PubMed1997

Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can Be functionally distinguished from the type I isoforms.

In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hydrodynamic parameters indicative of a homodimer. Chemical cross-linking studies showed that pig cGK II in intestinal membranes has a dimeric structure as well. However, after purification, cGK II was found to be partly proteolyzed into C-terminal monomeric fragments. Phosphorylation studies in rat intestinal brush borders revealed that the potency of cGMP analogs to stimulate or inhibit native cGK II in vitro (i.e. 8-(4-chlorophenylthio)-cGMP > cGMP > beta-phenyl-1,N2-etheno-8-bromo-cGMP > beta-phenyl-1,N2-etheno-cGMP and Rp-8-(4-chlorophenylthio)-cGMPs > Rp-beta-phenyl-1, N2-etheno-8-bromo-cGMPs, respectively) correlated well with their potency to stimulate or inhibit cGK II-mediated Cl- secretion across intestinal epithelium but differed strikingly from their potency to affect cGK I activity. These data show that the N terminus of cGK II is involved in dimerization and that endogenous cGK II displays a distinct activation/inhibition profile with respect to cGMP analogs, which permits a pharmacological dissection between cGK II- and cGK I-mediated physiological processes.

Animals↗

PubMed1997

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta.

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.

Adenoviridae↗

PubMed1997

Characterization of swelling-induced ion transport in HT-29Cl.19A cells. Role of inorganic and organic osmolytes during regulatory volume decrease.

Combined intracellular and transepithelial potential and resistance measurements were performed to localize the ion conductances activated by hypo-osmotic shock of cultured human colonic carcinoma cells (HT-29Cl.19A). Furthermore, the effect of cell swelling induced by a hypo-osmotic solution on the intracellular Ca2+ activity [Ca2+]i and release of amino acids into the extracellular solution was examined. Application of a 40% hypo-osmotic solution on both sides of confluent monolayers induced a hyperpolarization of the intracellular potential caused by increased K+ conductance of the basolateral membrane, followed by a sustained depolarization due to increased Cl- conductance in the apical and basolateral membranes. Usually no transepithelial current occurred, presumably because of random distribution of Cl- channels. However, in some monolayers cell swelling induced a transepithelial Cl- current because of a more pronounced expression of volume-sensitive Cl- channels in the apical membrane. Exposure to hypo-osmotic solution increased [Ca2+]i transiently. The increase of [Ca2+]i was also observed to occur in the presence of the muscarinic receptor agonist carbachol or the inhibitor of the microsomal Ca2+-ATPase thapsigargin (TG), which prevented carbachol-induced Ca2+ release, suggesting that cell swelling recruits Ca2+ from a different source compared to carbachol or TG. Following incubations with hypo-osmotic solutions, about 60% of the intracellular free amino acids including aspartate, glutamate, glycine and taurine was released. It is concluded that the regulatory volume decrease (RVD) in HT-29Cl.19A colonocytes is achieved by activation of K+ and Cl- conductances, resulting in net loss of salt, as well by extrusion of amino acids.

Amino Acids↗

PubMed1997

Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism.

Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.

3T3 Cells↗

PubMed1996

Hypo-osmotic cell swelling activates the p38 MAP kinase signalling cascade.

Hypo-osmotic swelling of human Intestine 407 cells leads to a significant increase of intracellular MAPKAP-kinase 2 activity and Hsp27 phosphorylation. Pre-treatment of the cells with the p38 MAP kinase inhibitor SB-203580 blocks this activation, indicating that the hypotonicity-induced activation of MAPKAP kinase 2 is, similarly to that described for hyperosmotic treatment, the result of an activated p38 MAP kinase cascade. The activation of MAPKAP kinase 2 proceeds with kinetics similar to that of one of the first physiological responses of hypo-osmotic treatment, the opening of compensatory Cl- channels. However, inhibition of the p38 MAP kinase cascade does not block the osmo-sensitive anion efflux and, vice versa, activation of p38 MAP kinase by cytokines and anisomycin does not increase the efflux. These results indicate that the p38 MAP kinase cascade is not directly involved in Cl- channel activation but instead may play a role in subsequent cellular repair processes.

Anisomycin↗

PubMed1996

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

Animals↗

PubMed1996

N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II.

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.

Animals↗

PubMed1996

Chloride and potassium conductances of mouse pancreatic zymogen granules are inversely regulated by a approximately 80-kDa mdr1a gene product.

Cl- and cation conductances were characterized in zymogen granules (ZG) isolated from the pancreas of wild-type mice (+/+) or mice with a homozygous disruption of the multidrug resistance P-glycoprotein gene mdr1a (-/-). Cl- conductance of ZG was assayed in isotonic KCl buffer by measuring osmotic lysis, which was induced by maximal permeabilization of ZG membranes (ZGM) for K+ with valinomycin due to influx of K+ through the artificial pathway and of Cl- through endogenous channels. To measure cation conductances, ZG (pHi 6.0-6.5) were suspended in buffered isotonic monovalent cation acetate solutions (pH 7.0). The pH gradient was converted into an outside-directed H+ diffusion potential by maximally increasing H+ conductance of ZGM with carbonyl cyanide m-chlorophenylhydrazone. Osmotic lysis of ZG was induced by H+ diffusion potential-driven influx of monovalent cations through endogenous channels and nonionic diffusion of the counterion acetate. ZGM Cl- conductances were not different in (-/-) and (+/+) mice (2.6 +/- 0.3 h-1 versus 3.1 +/- 0.2 h-1 (relative rate constant)). The nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (0.5 mM) activated the Cl- conductance both in (+/+) and (-/-) mice. However, activation of Cl- conductance by AMP-PCP was reduced in (-/-) mice as compared with (+/+) mice (5.0 +/- 0.4 h-1 versus 7.6 +/- 0.7 h-1; p < 0. 005). In contrast, ZGM K+ conductance was increased in (-/-) mice as compared with (+/+) mice (14.2 +/- 2.0 h-1 versus 8.5 +/- 1.2 h-1; p < 0.03). In the presence of 0.5 mm AMP-PCP, which completely blocks K+ conductance but leaves a nonselective cation conductance unaffected, there was no difference between (-/-) and (+/+) mice (5.3 +/- 0.7 h-1 versus 3.2 +/- 0.5 h-1). In Western blots of ZGM from wild-type mice, a polyclonal MDR1 specific antibody labeled a protein band of approximately 80 kDa. In mdr1a-deficient mice, the intensity of this band was reduced to 39 +/- 7% of the wild-type signal. This indicates that a mdr1a gene product of approximately 80 kDa enhances the AMP-PCP-activated fraction of mouse ZGM Cl- conductance and reduces AMP-PCP-sensitive K+ conductance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗