Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (alpha-, beta-, and gamma-subunits). To study the role of the beta-subunit in vivo, we analyzed mice in which the betaENaC gene locus was disrupted. These mice showed low levels of betaENaC mRNA expression in kidney (approximately 1%), lung (approximately 1%), and colon (approximately 4%). In homozygous mutant betaENaC mice, no betaENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult betaENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as betaENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, betaENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that betaENaC is required for Na+ conservation during salt deprivation.     

A novel sodium channel mutation causing a hyperkalemic paralytic and paramyotonic syndrome with variable clinical expressivity

BACKGROUND: The CarboMedics valve is a relatively new, low-profile, bileaflet, mechanical prosthesis. The results of a prospective follow-up study after valve replacement with this prosthesis in a university hospital are presented. METHODS: We implanted 640 CarboMedics prostheses in 583 patients in the aortic (n = 359), mitral (n = 167), or aortic and mitral positions (double valve replacement; n = 57). Patient ages ranged from 11 to 81 years (mean age, 58 +/- 12.3 years). RESULTS: Overall hospital mortality was 9.0%; however, when high-risk urgent cases were removed from the calculation, the operative mortality fell to 4.5%. Follow-up was 98% complete, comprising 2,027 patient-years for a mean follow-up of 44 months (range, 6 to 72 months). Actuarial freedom from complications (linearized rates in parentheses) was as follows: late mortality, 85% +/- 2.0% (2.3%/patient-year); thromboembolism, 92% +/- 1.1% (1.6%/patient-year); anticoagulation-related hemorrhage, 87% +/- 1.2% (2.8%/patient-year); prosthetic valve endocarditis, 98% +/- 0.5% (0.1%/patient-year); and overall valve-related morbidity and mortality, 76% +/- 2.1% (4.3%/ patient-year). CONCLUSIONS: The CarboMedics valve shows a low rate of valve-related complications comparable with other new mechanical heart valve prostheses.     

Novel GLRA1 missense mutation (P250T) in dominant hyperekplexia defines an intracellular determinant of glycine receptor channel gating

Missense mutations as well as a null allele of the human glycine receptor alpha1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A of GLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature alpha1 polypeptide. After recombinant expression, homomeric alpha1(P250T) subunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase in Ki values. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novel GLRA1 mutant allele P250T defines an intracellular determinant of glycine receptor channel gating.     

The startle disease mutation Q266H, in the second transmembrane domain of the human glycine receptor, impairs channel gating

Hyperekplexia (startle disease) results from mutations in the glycine receptor chloride channel that disrupt inhibitory synaptic transmission. The Q266H missense mutation is the only hyperekplexia mutation located in the transmembrane domains of the receptor. Using recombinant expression and patch-clamping techniques, we have investigated the functional properties of this mutation. The ability of glycine and taurine to open the channel was reduced in the mutated channel, as shown by a 6-fold shift in the concentration-response curve for both agonists. This was not accompanied by similar changes in agonist displacement of strychnine binding, suggesting that the mutation affects functions subsequent to ligand binding. Taurine was also converted to a weak partial agonist and antagonized the actions of glycine, consistent with changes in its channel gating efficacy. Because the Q266H mutation is within the pore-forming second transmembrane domain, we tested for a direct interaction with permeating ions. No change in either the cation/anion selectivity ratio or in single channel conductance levels was observed. No differential effects of Zn++, pH, and diethylpyrocarbonate were observed, implying that the histidine side chain is not exposed to the channel lumen. Single-channel recordings revealed a significant reduction in open times in the mutant receptors, at both high and low agonist concentrations, consistent with the open state of the channel being less stable. This study demonstrates that residues within the second transmembrane domain of ligand-gated ion channel receptors, even those whose side chains do not directly interact with permeating ions, can affect the kinetics of channel gating.     

One face of a transmembrane helix is crucial in mechanosensitive channel gating

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an alpha-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.     

Genetic analysis of the epithelial sodium channel in Liddle's syndrome

BACKGROUND: Liddle's syndrome is an autosomal inheritable disorder that causes hypertension due to excess function of sodium channel. OBJECTIVE: To analyze the DNA sequence of the amiloride-sensitive epithelial sodium channel (ENaC) in three patients who had low-renin hypertension with hypokalemia. The patients included a 24-year-old woman and her 20-year-old brother whose mother was hypertensive. The third patient was a 15-year-old girl with no family history of hypertension. METHODS: The DNA sequence of the ENaC was analyzed as follows. Venous blood samples were collected from the patients and total genomic DNA was prepared by standard methods. Specific primers were used for direct polymerase chain reaction; one set of primers for amplifying the C terminus (codon 523-638) of the , subunit of ENaC, and two sets of primers for amplifying the C terminus (codons 525-587 and 568-650) of the y subunit of ENaC. Polymerase chain reaction products were purified and subjected to direct DNA sequence analysis. RESULTS: Direct sequence analysis demonstrated the presence of a single-base substitution in one segment of the 0 subunit of ENaC, a C-T transition that changed the encoded Pro (CCC) at codon 616 to Ser (TCC) in the siblings (cases 1 and 2). In case 3, we found a missense mutation of Pro (CCC) to Leu (CTC) at codon 616. Case 3 is considered to be sporadic, since DNA sequencing of the PY motif of her parents gave normal results. CONCLUSIONS: The DNA sequences of the ENaC in three patients with Liddle's syndrome were analyzed. In one family case, we found a new missense mutation of Pro (CCC) to Ser (TCC) at codon 616 in the 0 subunit of ENaC. A genetic analysis of the amiloride-sensitive epithelial sodium channel is recommended in assessing patients with low-renin, salt-sensitive hypertension whose blood pressure is not responsive to spironolactone treatment.     

A Japanese family with paramyotonia congenita which has a mutation in the muscle sodium channel gene

We report here a Japanese family with paramyotonia congenita. The proband was a 42-year-old woman (case 1), who noticed muscle stiffness and weakness in the cold since the age of 7 years. These symptoms were alleviated by warming. Her eldest son (case 2) also experienced similar symptoms, while her younger son and daughter were healthy. Neurological examination in case 1 revealed mild weakness in facial and neck muscles. Cold-induced muscle stiffness and weakness were present. Electromyography showed myotonic discharges, intensified by cooling or repetitive exercise. The amplitude of the compound muscle action potentials was also reduced by the repetitive exercise and cooling. Serum chemistry including potassium and CK was normal. Molecular analysis of SCN4A (exon22-24) by SSCP and nucleotide sequencing revealed a C-to-T transition at nucleotide 3,938, causing a substitution of 1313methionine of threonine in case 1. This mutation was confirmed by PCR-RFLP with a mismatched primer; the proband (case 1) and her eldest son (case 2) had a heterozygous mutation, while the other family members did not. This is the first report that a mutation in SCN4A was identified in a Japanese family with paramyotonia congenita.     

A sec1-related vesicle-transport protein that is expressed predominantly in epithelial cells

Sec1-related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Sec1-related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc-18-2/Munc-18b/muSec1 protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associates in vitro with syntaxins 1A, 2, and 3. In situ hybridization analysis of embryonic mouse tissues revealed prominent expression of the munc-18-2 mRNA in the epithelia of several tissues. Cell-fractionation studies demonstrated that the majority of Munc-18-2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc-18-2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS-1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin 1A, it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc-18-2 as a predominantly epithelial vesicle-transport protein with a polarized distribution and provided novel in vivo evidence for the association of Sec1-related proteins with members of the syntaxin family.     

Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype

The epithelial Na+ channel (ENaC) is composed of three homologous subunits: alpha, beta and gamma. We used gene targeting to disrupt the beta subunit gene of ENaC in mice. The betaENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the -/- mice, we found an increased urine Na+ concentration despite hyponatremia and a decreased urine K+ concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to alphaENaC-deficient mice, which die because of defective lung liquid clearance, neonatal betaENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo, the beta subunit is required for ENaC function in the renal collecting duct, but, in contrast to the alpha subunit, the beta subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the betaENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.     

Characterization of a natural mutation in an antigenic site on the fusion protein of measles virus that is involved in neutralization

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R-->W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.     

Mineralocorticoid hormone receptor and the epithelial sodium channel in a human leukemic cell line

A Cell extract from the HEL (human erythroblastic leukemia) cell line was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as glycosylated 82-84 kDa bands, and a single 102 kDa band, respectively, in Western blots using polyclonal antibodies raised against these proteins. The immunofluorescent labeling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor whereas the ENaC was exclusively membrane-bound. These results were confirmed by confocal microscopy. The expression of the MCR in HEL cells was evident as a predicted band of 843 bp (234 amino acids) after total RNA from HEL cells had been reverse transcribed and then amplified by PCR; the ENaC was similarly evident as a predicted band of 520 bp. In both cases, near 100% identity was observed between the deduced amino acid sequences of the PCR products and those from known human sources. The multiplication of HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population.     

A GLRA1 null mutation in recessive hyperekplexia challenges the functional role of glycine receptors

Dominant missense mutations in the human glycine receptor (GlyR) alpha 1 subunit gene (GLRA1) give rise to hereditary hyperekplexia. These mutations impair agonist affinities and change conductance states of expressed mutant channels, resulting in a partial loss of function. In a recessive case of hyperekplexia, we found a deletion of exons 1-6 of the GLRA1 gene. Born to consanguineous parents, the affected child is homozygous for this GLRA1(null) allele consistent with a complete loss of gene function. The child displayed exaggerated startle responses and pronounced head-retraction jerks reflecting a disinhibition of vestigial brain-stem reflexes. In contrast, proprio- and exteroceptive inhibition of muscle activity previously correlated to glycinergic mechanisms were not affected. This case demonstrates that, in contrast to the lethal effect of a null allele in the recessive mouse mutant oscillator (Glra1 spd-ot), the loss of the GlyR alpha 1 subunit is effectively compensated in man.     

A protein phosphatase is involved in the inhibition of histone deacetylation by sodium butyrate

Treatment of cells with sodium butyrate is known to increase histone acetylation by inhibiting deacetylases. Here we have observed, in cultured hepatoma cells, that the potent serine-threonine phosphatase inhibitors, okadaic acid or calyculin A, inhibited phosphatase activity and concomitantly decreased the histone acetylation classically maintained by sodium butyrate. These results suggest that a protein phosphatase may mediate the sodium butyrate effect on deacetylases. Since we have previously found that such a protein would also mediate the sodium butyrate effect on gene expression, we propose that a phosphatase activity constitutes an early and essential step in the sodium butyrate-triggered signalling pathway.     

A 50-picosiemens anion channel of the chloroplast envelope is involved in chloroplast protein import

Single channel recordings were used to investigate the changes on the pea chloroplast envelope during protein import. In the inside-out patch configuration a 50-picosiemens (pS) anion channel of the chloroplast envelope membrane was identified. The open time probability of the channel was decreased by the addition of the wild type precursor protein of ferredoxin (wt-prefd) to the pipette-filling solution in the presence of 0.5 mM ATP. In the absence of ATP or in the presence of 50 microM ATP, wt-prefd did not affect the open time probability of the channel. A deletion mutant of prefd, Delta6-14-prefd, which is inactive in in vitro import, was also unable to affect the open time probability of the 50-pS anion channel. In the presence of 100 microM ATP, wt-prefd decreased the open time probability of the channel to a lesser extent, as did the transit peptide alone. It is concluded that the 50-pS anion channel could be part of the protein import machinery of the inner membrane. In addition the precursor protein under import conditions induced burst-like increases of the envelope conductivity. The implication of both responses for the chloroplast protein import process are discussed.     

Membrane type 1-matrix metalloproteinase is involved in the formation of hepatocyte growth factor/scatter factor-induced branching tubules in madin-darby canine kidney epithelial cells

The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell cycle and a potential tumor suppressor gene. Because we had previously demonstrated that loss of p27 protein is associated with aggressive behavior in colorectal adenocarcinomas, we used immunohistochemistry and in situ hybridization to evaluate the potential role of alterations in p27 expression in primary and metastatic colorectal adenocarcinomas. Parallel immunostaining was performed for Ki-67 and p53. We evaluated 13 cases of metachronous and 23 cases of synchronous primary and metastatic colorectal tumor pairs. In the synchronous subgroup (Stage IV tumors), 57% of the primary tumor and metastases pairs did not express p27 protein and the remainder were low expressors. In the metachronous subgroup, 54% of the primary tumors were low expressors and the remainder high expressors of p27 protein. There was a significant reduction in the expression of p27 in the metachronous metastases (mean positive cells: 14.5%) when compared to the corresponding primary tumors (mean positive cells: 41.8%), P = 0.0023. All the primary and metastatic tumors in the metachronous subgroup showed high levels of p27 mRNA expression. There was no association between loss of p27 and either Ki-67 count or p53 expression. Because p27 is known to be up-regulated when epithelial cells are grown in suspension, the down-regulation of p27 in circulating tumor cells may confer the ability to grow in an environment of altered extracellular matrix or intercellular adhesion properties, two situations which may facilitate metastases.     

A mutation in the aryl hydrocarbon receptor (AHR) in a cultured mammalian cell line identifies a novel region of AHR that affects DNA binding

Introduction of a retroviral expression vector for the aryl hydrocarbon receptor (AHR) restores CYP1A1 inducibility to a mutant derivative of the Hepa-1 cell line that is defective in induction of CYP1A1 by ligands for the receptor. An AHR protein with normal ligand binding activity is expressed in the mutant but ligand treatment of mutant cell extract fails to induce binding of the AHR. ARNT (aryl hydrocarbon receptor nuclear translocator) dimer to the xenobiotic responsive element (XRE). AHR cDNAs derived from the mutant encode a protein that is unimpaired in ligand-dependent dimerization with ARNT, but the AHR.ARNT dimer so formed is severely impaired in XRE binding activity. The mutant cDNAs contain a C to G mutation at base 648, causing a cysteine to tryptophan alteration at amino acid 216, located between the PER-ARNT-SIM homology region (PAS) A and PAS B repeats. Introduction of the same mutation in the wild-type AHR sequence by site-directed mutagenesis similarity impaired XRE binding activity. Substitution with the conservative amino acid, serine, had no effect on XRE binding. The tryptophan mutation, but not the wild-type allele, was detectable in genomic DNA of the mutant. The implication that an amino acid within the PAS region may be involved in DNA binding indicates that the DNA binding behavior of AHR may be more anomalous than previously suspected.     

Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non-targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the 'post-P loop' (PP) regions. These PP mutations specifically caused marked defects in the 'C1' states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non-targeted mutagenesis and microbial selection can be a valuable tool for probing structure-function relationships of ion channels.     

Identification of a domain of lecithin-cholesterol acyltransferase that is involved in interfacial recognition

Lecithin-cholesterol acyltransferase (LCAT) is an interfacial enzyme that acts on lipid substrates on the surface of high density lipoproteins (HDL). Based on observations with other interfacial lipases, we propose that LCAT contains a surface region of 25 amino acids linked by a disulfide bond (C50-C74) that is involved in the binding of LCAT to lipoproteins. Using LCAT cDNA, we have deleted most of this region (delta 53-71) and expressed the mutant enzyme (LCAT delta 53-71) in COS-1 cells. The deletion mutant is expressed and secreted at levels similar to wildtype LCAT, suggesting that the deleted region is located on the surface of the enzyme and is not required for folding. The enzymatic activity of the mutant was tested using two interfacial substrates, reconstituted HDL (rHDL) and low density lipoprotein (LDL), as well as a water soluble substrate, p-nitrophenyl butyrate (PNPB). There was no reaction with rHDL and LDL, but 30% of the activity with PNPB was retained. This suggests that the deleted region plays a role in interfacial binding, while the active site core is not disrupted. We thus conclude that this region (C50-C74) forms part of the interfacial binding domain of LCAT.