Biomedical subjects

W B Guggino

Publications and source records attributed to W B Guggino.

At least 91 records · Page 5Linked to original sources

PubMed1995

CIC-2: a developmentally dependent chloride channel expressed in the fetal lung and downregulated after birth.

Growth and differentiation of the fetal lung are dependent on chloride and fluid secretion, yet the specific molecular identities of fetal chloride channels have not been fully determined. In this study, we demonstrate mRNA expression of the volume-activated chloride channel, CIC-2, in fetal rat lung using reverse-transcriptase polymerase chain reaction (RT-PCR) and ribonuclease (RNase) protection assay. By RNase protection assay, CIC-2 mRNA expression is most abundant in fetal lung and diminishes after birth until it is almost undetectable in adult rat lung. To confirm this result at the protein level, a C-terminal fragment of CIC-2 cDNA derived from 19-day fetal rat lung was cloned into an expression plasmid. The truncated 33-kD CIC-2 protein was expressed in Escherichia coli and purified by column chromatography. Polyclonal antibodies to this antigen were raised in chickens, and the antisera detected a 94-kD protein in fetal rat lung homogenates by Western blotting. Protein expression of CIC-2 was most abundant in mid and late gestation and decreased significantly shortly after birth, as would be predicted by the RNase protection data. CIC-2 protein was localized along the apical surface of fetal airway epithelium by immunocytochemistry. The abundant fetal expression of CIC-2 RNA and protein supports the hypothesis that CIC-2 is important to fetal lung development, and its apical location suggests that it may be involved in fluid secretion during normal lung morphogenesis.

Animals↗

PubMed1995

A single histidine residue is essential for zinc inhibition of GABA rho 1 receptors.

The GABA rho 1 subunit, cloned from a human retina library, can form homooligomeric receptors with properties similar to GABAc receptors characterized in retinal cells. The divalent cation Zn2+, abundant in the CNS and retina, was found to inhibit GABA rho 1 receptors in a voltage-independent manner. Varying the extracellular pH from 7.4 to 5.6 significantly reduced this inhibitory effect. This pH profile suggested that one or more histidine residues might play a role in the interaction between Zn2+ and the GABA rho 1 receptor. Site-directed mutagenesis revealed that a single histidine residue (His 156) in the putative extracellular domain of rho 1 was critical for Zn2+ sensitivity. Substitution of this amino acid with tyrosine (H156Y) created a functional GABA receptor with agonist and channel properties indistinguishable from wildtype. However, the H156Y mutant was insensitive to Zn2+, even at concentrations as high as 1 mM. Mutation to aspartic acid, an amino acid that can interact with Zn2+ in other proteins, preserved sensitivity to Zn2+ but abolished the pH-dependent effect. This histidine residue is also involved in Ni2+ and Cd2+ interaction since the H156Y mutation completely suppressed the inhibition effects of these two cations. These data demonstrate that an extracellular histidine residue is critical for transition metal cation sensitivity of GABA rho 1 receptors.

Animals↗

PubMed1995

Alternatively spliced forms of the alpha subunit of the epithelial sodium channel: distinct sites for amiloride binding and channel pore.

The amiloride-sensitive epithelial sodium channel (ENAC) consists of at least three subunits, alpha, beta, and gamma. Sodium conductance occurs when only the alpha subunit is expressed in Xenopus oocytes, but it is greatly enhanced by coexpression of all three subunits. All three subunits have two transmembrane domains. Whether the amiloride binding site exists in the extracellular portion or a transmembrane domain has not been established. Using reverse transcription-polymerase chain reaction in rat taste tissues, we have identified two alternatively spliced transcripts of ENAC (alpha ENACa and alpha ENACb) with deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxyl terminus. Genomic Southern blots indicate that a single gene accounts for alpha ENAC and the alternatively spliced variants. Reverse transcription-polymerase chain reaction and RNase protection assays demonstrate that alpha ENACa is expressed to a lesser extent than alpha ENAC in kidney, lung, and taste tissues. alpha ENACa differs from alpha ENAC by a deletion in the second transmembrane domain. Despite this deletion, alpha ENACa expression in transfected human embryonic kidney 293 cells or CV-1 cells augments [3H]phenamil binding. The [3H]phenamil binding of alpha ENACa resembles that of alpha ENAC, being inhibited more potently by phenamil (Kd = 65 nM) than amiloride. Unlike alpha ENAC, expression of alpha ENACa in Xenopus oocytes fails to generate amiloride-sensitive Na+ or Li+ currents. These results suggest that the amiloride binding site resides on the extracellular loop of the alpha subunit of ENAC and not the putative second transmembrane domain, which forms a channel pore. Heterogeneity in alpha ENAC isoforms may contribute to the complexity of multimeric structures and functional variation of ENAC.

Alternative Splicing↗

PubMed1995

An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction.

Adeno-associated virus (AAV) vectors are potentially useful for gene therapy of a number of human diseases. However, the use of these vectors has been limited by the lack of stable vector-packaging cell lines. The difficulties in developing packaging cell lines relate to low levels of rep gene expression from the AAV-p5 promoter, and to the propensity of Rep proteins to suppress continued growth of immortalized cell lines. We describe here two new techniques which allow these problems to be circumvented. First, we have demonstrated that expression of rep from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter results in a 10-fold improvement of packaging efficiency. Second, we have overcome the inefficiency of vector plasmid transfection by generating cell populations containing rescuable AAV recombinant genomes. These improvements yielded a net increase of 50-fold in the packaging efficiency of the AAVp5neo and AAVp5lacZ recombinant vectors. The AAVp5lacZ vector packaged with this method was administered systemically to recently weaned C57BL mice, and mediated efficient expression of the beta-galactosidase reporter gene in cells of the airway epithelium and spleen. This indicates the in vivo activity of these vector stocks, and their potential utility for gene therapy.

Animals↗

PubMed1995

Alveolar stem cell transduction by an adeno-associated viral vector.

In inherited disorders such as surfactant protein deficiencies or cystic fibrosis (CF), where lung damage develops progressively after birth, gene replacement is best accomplished in the neonatal period. We use the adeno-associated virus (AAV) as a vector for gene transfer in the newborn rabbit lung where stem cells are activated for lung growth and differentiation. AAV-mediated gene transfer as assayed by lacZ gene expression occurred preferentially in alveoli in the alveolar epithelial progenitor cell, the type II cell, and in the large airway tracheobronchial basal and ciliated cells. Cell proliferation was confirmed by 5-bromo-deoxyuridine (BRDU) labeling in regions undergoing alveolarization and airway branch points. Regions of cell proliferation coincided with areas of significant lacZ expression. Thus, dividing and differentiating cells can be targeted by AAVlacZ delivery to newborn lung.

Animals↗

PubMed1994

Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance.

The aquaporins transport water through membranes of numerous tissues, but the molecular mechanisms for sensing changes in extracellular osmolality and regulating water balance in brain are unknown. We have isolated a brain aquaporin by homology cloning. Like aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa), the deduced polypeptide has six putative transmembrane domains but lacks cysteines at the known mercury-sensitive sites. Two initiation sites were identified encoding polypeptides of 301 and 323 amino acids; expression of each in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability not blocked by 1 mM HgCl2, even after substitution of cysteine at the predicted mercury-sensitive site. Northern analysis and RNase protection demonstrated the mRNA to be abundant in mature rat brain but only weakly detectable in eye, kidney, intestine, and lung. In situ hybridization of brain localized the mRNA to ependymal cells lining the aqueduct, glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of hypothalamus, and Purkinje cells of cerebellum. Its distinctive expression pattern implicates this fourth mammalian member of the aquaporin water channel family (designated gene symbol, AQP4) as the osmoreceptor which regulates body water balance and mediates water flow within the central nervous system.

Amino Acid Sequence↗

PubMed1994

Molecular structure of the water channel through aquaporin CHIP. The hourglass model.

Aquaporin channel-forming integral protein (CHIP) is the first characterized water channel protein (genome symbol AQP1), but the molecular structure of the aqueous pathway through CHIP remains undefined. The two halves of CHIP are sequence-related, and each has three bilayer-spanning domains with the motif asparagine-proline-alanine (NPA) at residues 76-78 (in cytoplasmic loop B) and 192-194 (in extracellular loop E). The NPA motifs are oriented 180 degrees to each other, and the second NPA is near cysteine 189, the known site where mercurials inhibit osmotic water permeability (Pf). When expressed in Xenopus oocytes, the double mutant A73C/C189S exhibited high, mercurial-sensitive Pf similar to wild-type CHIP. Conservative substitutions of slightly greater mass in or near NPA motifs in loop B or loop E in CHIP caused reduced Pf and failure of the protein to localize at the plasma membrane. Certain nonfunctional loop E mutants complemented the truncation mutant D237Z. Formation of mixed oligomers was demonstrated by velocity sedimentation, immunoprecipitation, and analysis of dimeric-CHIP polypeptides. Cellular distributions of individual mutants or complementing pairs of mutants were verified by plasma membrane isolation and confocal microscopy. An hourglass structural model is proposed in which a cytoplasmic chamber (loop B) connects within the membrane to an extracellular chamber (loop E) forming a single, narrow aqueous pathway through each of the CHIP subunits; subunit oligomerization may provide the vertical symmetry necessary for residence within the lipid bilayer.

Amino Acid Sequence↗

PubMed1994

Membrane topology of aquaporin CHIP. Analysis of functional epitope-scanning mutants by vectorial proteolysis.

CHIP is the archetypal member of the aquaporins, a widely expressed family of membrane water channels. The NH2- and COOH-terminal halves of CHIP are sequence-related, and hydropathy analysis predicted six membrane-spanning domains with five connecting loops (A-E). Here, we determined the membrane topology of CHIP expressed in Xenopus oocytes using biologically active recombinant channels. CHIP is glycosylated at Asn-42, indicating loop A is exofacial. An epitope from the coronavirus E1 glycoprotein was inserted into CHIP and localized to the outer or inner leaflet of the membrane by alpha-chymotrypsin digestion of intact oocytes or inside-out membrane vesicles. The E1 epitope at Thr-120 was protease-sensitive in intact oocytes, indicating that loop C is exofacial. The E1 epitope at Lys-6, Arg-162, or Lys-267 was protease-sensitive in inside-out membrane vesicles, confirming the cytoplasmic location of the NH2 and COOH termini and loop D. Insertions into loops B and E did not produce active water channels, but their cleavage patterns were consistent with inner (loop B) and outer (loop E) leaflet locations. This study indicates that the functional CHIP molecule is a unique structure with two internal repeats oriented 180 degrees to each other within the membrane.

Amino Acid Sequence↗

PubMed1994

Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator.

BACKGROUND/AIMS: Human rectal epithelium in cystic fibrosis (CF) shows impaired ion transport in response to theophylline or bethanechol, although it possesses regulatory subunits of adenosine cyclic 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A). Protein kinase A-specific phosphorylation of CF transmembrane conductance regulator (CFTR) in rectal tissues of control and CF volunteers was examined in this study. METHODS: CFTR was evaluated using a polyclonal antiserum (pre-NBF) raised against a peptide corresponding to residues 415-427 of CFTR. Microsomal membranes from normal and CF rectal mucosa and from T-84 cells were incubated with [gamma 32P]-adenosine triphosphate +/- protein kinase A and subjected to immunoblotting with pre-NBF and autoradiography. RESULTS: Pre-NBF recognized a single band of 180 kilodaltons. Protein kinase A altered phosphorylation of this 180-kilodalton band 1.4-, 2.2- and 0.9-fold in T-84, normal, and CF rectal membranes, respectively. Catalytic activities of protein kinase A, Ca2+ calmodulin protein kinase, or protein kinase C in control and CF tissues were similar. CONCLUSIONS: cAMP and Ca(2+)-signaling pathways are normal up to the kinases in CF rectal mucosa. Our results suggest differences in CFTR phosphorylation in normal and CF rectal mucosal membranes.

Adult↗

PubMed1994

Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents.

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

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

PubMed1994

A novel gamma-aminobutyric acid receptor subunit (rho 2) cloned from human retina forms bicuculline-insensitive homooligomeric receptors in Xenopus oocytes.

The rho 2 subunit, a novel GABA receptor subunit, has been cloned from a human retinal complementary DNA library. This subunit shares 74% amino acid sequence identity with the rho 1 subunit that forms homooligomeric bicuculline-, barbiturate-, and benzodiazepine-insensitive GABA receptors. The rho 2 subunit also forms homooligomeric GABA-activated chloride channels when expressed in Xenopus oocytes. The amplitudes of the whole-cell currents of rho 2 receptors are always smaller than those of rho 1 receptors, but the affinity and cooperativity of GABA are very similar. Like the rho 1 subunit, the currents generated by rho 2 are insensitive to GABAA receptor modulators including bicuculline, hexobarbital, and diazepam and can be reversibly inhibited by ZnCl2. Homooligomeric rho 2 and rho 1 receptors are less sensitive to muscimol and picrotoxin, and desensitize slower than GABAA receptors. These data demonstrate that homooligomeric receptors formed by rho 2 and rho 1 subunits have a number of electrophysiologic and pharmacologic characteristics that differ from receptors formed by GABAA receptor subunits. The distinctive properties of rho receptors are very similar to those of bicuculline-insensitive GABA-gated chloride channels identified in retina, suggesting a molecular basis for this form of GABA receptor in visual pathways.

Animals↗

PubMed1993

Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.

Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.

Amino Acid Sequence↗

PubMed1993

GABA rho 2 receptor pharmacological profile: GABA recognition site similarities to rho 1.

The rho 2 receptor for gamma-aminobutyric acid (GABA) induces GABA-gated currents when expressed as a homooligomer in Xenopus oocytes. rho 2 responses display pharmacological profiles similar to those of expressed rho 1 receptors, although responses were slower, most agonists were more potent at rho 2 and Im values for the partial agonist imidazole-4-acetic acid were 7-fold higher. Amino acids important for most aspects of GABA agonist ligand recognition may not be among those that differ between rho 1 and rho 2, including the 20% amino acid difference in the N-terminal region.

Animals↗

PubMed1993

Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.

Adeno-associated virus type 2 (AAV) vectors have been used for gene expression in respiratory epithelial cells and may be useful in gene therapy for diseases like cystic fibrosis (CF) which affect the airways. The AAV p5 promoter together with the AAV inverted terminal repeat (ITR) forms a 263-base pair cassette which mediated efficient expression in a CF bronchial epithelial cell line. We report here that the ITR itself can mediate gene expression. In stable transfection assays, AAV-CF vectors expressing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA from either the p5 promoter or the ITR restored cAMP regulation of the chloride efflux characteristic of CFTR function. An AAV-ITR-CF vector deleted for the amino terminus of CFTR was also functional. This vector was packaged into AAV particles and used to transduce cells without selection. Transduced cells also exhibited cAMP-regulated Cl- efflux. The complemented cell lines showed increased levels of CFTR protein immunofluorescence, and the presence of intact AAV-CF vector sequence was confirmed by Southern blot analysis of rescued vector sequences. These studies provide novel insights into AAV gene expression, and this newly described promoter allows for the production of AAV vectors expressing CFTR in those differentiated cells affected in CF.

Base Sequence↗

PubMed1993

The mercury-sensitive residue at cysteine 189 in the CHIP28 water channel.

Water channels provide the plasma membranes of red cells and renal proximal tubules with high permeability to water, thereby permitting water to move in the direction of an osmotic gradient. Molecular identification of CHIP28 protein as the membrane water channel was first accomplished by measurement of osmotic swelling of Xenopus oocytes injected with CHIP28 RNA (Preston, G.M., Carroll, T.P., Guggino, W.B., and Agre, P. (1992) Science 256, 385-387). Since water channels are pharmacologically inhibited by submillimolar concentrations of Hg2+, site-directed mutagenesis was undertaken to demonstrate which of the 4 cysteines (87, 102, 152, or 189) is the Hg(2+)-sensitive residue in the CHIP28 molecule. Each cysteine was individually replaced by serine, and oocytes expressing each of the four mutants exhibited osmotic water permeability (Pf) equivalent to wild-type CHIP28. After incubation in HgCl2, all were significantly inhibited, except C189S exists as a multisubunit complex in the native membrane; however, although oocytes injected with mixed CHIP28 and C189S RNAs exhibited Pf corresponding to the sum of their individual activities, exposure to Hg2+ only reduced the Pf to the level of the C189S mutant. Of the six substitutions at residue 189, only the serine and alanine mutants exhibited increased Pf and had glycosylation patterns resembling wild-type CHIP28 on immunoblots. These studies demonstrated: (i) CHIP28 water channel activity is retained despite substitution of individual cysteines with serine; (ii) cysteine 189 is the Hg(2+)-sensitive residue; (iii) the subunits of the CHIP28 complex are individually active water pores; (iv) residue 189 is critical to proper processing of the CHIP28 protein.

Amino Acid Sequence↗

PubMed1993

Identification and regulation of whole-cell Cl- and Ca(2+)-activated K+ currents in cultured medullary thick ascending limb cells.

The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca(2+)-activated K+ current was characterized by its voltage-dependent and Ca(2+)-dependent properties. When the extracellular K+ ion concentration was increased from 2 to 140 mM, the re-reversal potential (Ek) was shifted from -85 to 0 mV with a slope of 46 mV per e-fold change. The Ca(2+)-activated K+ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca(2+)-activated K+ channel studied with the single channel patch-clamp technique. The results suggest that the Ca(2+)-activated K+ current is the predominant, large conductance and Ca(2+)-dependent K+ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl- current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I-V) relationship of the Cl- current showed an outward-rectifying pattern in symmetrical Cl- solution. The Cl- selectivity of the whole-cell current was confirmed by tail current analysis in different Cl- concentration bath solutions. Several Cl- channel blockers were found to be effective in blocking the outward-rectifying Cl- current in MTAL cells. The cAMP-dependent Cl- transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl- sensitive dye using fluorescence microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1993

Alterations in a voltage-gated K+ current during the differentiation of ML-1 human myeloblastic leukemia cells.

A voltage-gated K+ current has been identified in ML-1 human myeloid leukemia cells, with the use of the whole-cell patch-clamp technique. ML-1 cells proliferate in tissue culture as immature myeloblasts and can be induced to differentiate to nonproliferative monocyte/macrophages. In the myeloblastic cells, activation of the K+ current occurs upon depolarization of the membrane potential to above -40 mV; inactivation of this current is also voltage dependent and follows a simple exponential time course with a time constant (Ti) of 900 msec at 0 mV. The current is inhibited by 4-aminopyridine (IC50 of 80 microM at 0 mV), but is much less sensitive to tetraethylammonium of Ba2+. In cells exposed to the differentiation-inducer 12-O-tetradecanoylphorbol-13-acetate (TPA), dramatic alterations in the K+ current occur: upon exposure to 10 nM TPA during whole-cell recording, the amplitude of the voltage-activated current initially increases (within 4 min) and later decreases (at approximately 30-50 min). Upon addition of 0.5 nM TPA to cells in tissue culture, the current shows suppressed activation and accelerated inactivation in the early stages of differentiation (10-fold decrease in Ti at approximately 7 hr) and is completely suppressed in the later stages (3 days). Thus, this voltage-gated K+ current is suppressed early in the induction of differentiation and associated loss of proliferation in myeloid ML-1 cells exposed to TPA; this parallels the fact that channels of a similar type are activated upon the stimulation of proliferation in lymphoid cells exposed to mitogens.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗

PubMed1993

Outwardly rectifying chloride channels and CF: a divorce and remarriage.

Outwardly rectifying Cl- channels were originally thought to be the central element in cystic fibrosis. The role of these channels in CF was questioned to such an extent that doubts were raised about the validity of the original experiments. Recent data reestablishes a role for outwardly rectifying Cl- channels (ORCC) in CF and suggests that the protein encoded by the CF gene, the cystic fibrosis transmembrane regulator (CFTR), can effect the regulation of more than one channel in the airway. This minireview deals with the rise, fall, and resurrection of the role of outwardly rectifying Cl- channels in CF.

Chloride Channels↗