Genetic heterogeneity in Leigh syndrome: Highlighting treatable and novel genetic causes.

Leigh syndrome (LS), the most common childhood mitochondrial disorder, has characteristic clinical and neuroradiologic features. Mutations in more than 75 genes have been identified in both the mitochondrial and nuclear genome, implicating a high degree of genetic heterogeneity in LS. To profile these genetic signatures and understand the pathophysiology of LS, we recruited 64 patients from 62 families who were clinically diagnosed with LS at Seoul National University Children's Hospital. Mitochondrial genetic analysis followed by whole-exome sequencing was performed on 61 patients. Pathogenic variants in mitochondrial DNA were identified in 18 families and nuclear DNA mutations in 22. The following 17 genes analyzed in 40 families were found to have genetic complexity: MTATP6, MTND1, MTND3, MTND5, MTND6, MTTK, NDUFS1, NDUFV1, NDUFAF6, SURF1, SLC19A3, ECHS1, PNPT1, IARS2, NARS2, VPS13D, and NAXE. Two treatable cases had biotin-thiamine responsive basal ganglia disease, and another three were identified as having defects in the newly recognized genes (VPS13D or NAXE). Variants in the nuclear genes that encoded mitochondrial aminoacyl tRNA synthetases were present in 27.3% of cases. Our findings expand the genetic and clinical spectrum of LS, showing genetic heterogeneity and highlighting treatable cases and those with novel genetic causes.

Importance of early diagnosis in LMNA-related muscular dystrophy for cardiac surveillance.

INTRODUCTION: The identification of LMNA-related muscular dystrophy is important because it poses life-threatening cardiac complications. However, diagnosis of LMNA-related muscular dystrophy based on clinical features is challenging. METHODS: We reviewed the clinical phenotypes of 14 children with LMNA variants, focusing on the cardiac function and genotypes. RESULTS: Most patients presented with motor developmental delay or gait abnormalities. Eight (57%) patients had prominent neck extensor weakness or contractures. All patients showed ankle contractures at an early stage. Regular cardiac surveillance allowed for the detection of dysrhythmias in 57% of patients at a mean age of 14 years (range, 5-26). All patients had missense variants; however, there were no clear phenotype-genotype correlations. DISCUSSION: Early diagnosis of LMNA-related muscular dystrophy provides an opportunity for cardiac surveillance, potentially leading to the prevention of cardiac mortality in children.

GABBR2 mutations determine phenotype in rett syndrome and epileptic encephalopathy.

OBJECTIVE: Rett syndrome (RTT) and epileptic encephalopathy (EE) are devastating neurodevelopmental disorders with distinct diagnostic criteria. However, highly heterogeneous and overlapping clinical features often allocate patients into the boundary of the two conditions, complicating accurate diagnosis and appropriate medical interventions. Therefore, we investigated the specific molecular mechanism that allows an understanding of the pathogenesis and relationship of these two conditions. METHODS: We screened novel genetic factors from 34 RTT-like patients without MECP2 mutations, which account for ∼90% of RTT cases, by whole-exome sequencing. The biological function of the discovered variants was assessed in cell culture and Xenopus tropicalis models. RESULTS: We identified a recurring de novo variant in GABAB receptor R2 (GABBR2) that reduces the receptor function, whereas different GABBR2 variants in EE patients possess a more profound effect in reducing receptor activity and are more responsive to agonist rescue in an animal model. INTERPRETATION: GABBR2 is a genetic factor that determines RTT- or EE-like phenotype expression depending on the variant positions. GABBR2-mediated γ-aminobutyric acid signaling is a crucial factor in determining the severity and nature of neurodevelopmental phenotypes. Ann Neurol 2017;82:466-478.

Collagen VI-related myopathy: Expanding the clinical and genetic spectrum.

INTRODUCTION: We aimed to analyze the clinical and genetic characteristics of collagen VI-related myopathy. METHODS: We analyzed the clinical course and mutation spectrum in patients with collagen VI gene mutations among our congenital muscular dystrophy cohort. RESULTS: Among 24 patients with mutations in collagen VI coding genes, 13 (54.2%) were categorized as Ullrich type, and 11 (45.8%) as non-Ullrich type. Congenital orthopedic problems were similarly observed in both types, yet multiple joint contractures were found only in the Ullrich type. Clinical courses and pathology findings varied between patients. Mutations in COL6A1, COL6A2, and COL6A3 were found in 15 (65%), 3 (13%), and 5 (22%) patients, respectively, without genotype-phenotype association. Five novel variants were detected. DISCUSSION: We verified clinical heterogeneity of collagen VI-related myopathy, which emphasizes the importance of genetic testing. Genotype-phenotype association or early predictors for progression were not identified. Multiple joint contractures predict rapid deterioration. Muscle Nerve 58: 381-388, 2018.

Consecutive analysis of mutation spectrum in the dystrophin gene of 507 Korean boys with Duchenne/Becker muscular dystrophy in a single center.

INTRODUCTION: Duchenne and Becker muscular dystrophies (DMD and BMD) are allelic X-linked recessive muscle diseases caused by mutations in the large and complex dystrophin gene. METHODS: We analyzed the dystrophin gene in 507 Korean DMD/BMD patients by multiple ligation-dependent probe amplification and direct sequencing. RESULTS: Overall, 117 different deletions, 48 duplications, and 90 pathogenic sequence variations, including 30 novel variations, were identified. Deletions and duplications accounted for 65.4% and 13.3% of Korean dystrophinopathy, respectively, suggesting that the incidence of large rearrangements in dystrophin is similar among different ethnic groups. We also detected sequence variations in >100 probands. The small variations were dispersed across the whole gene, and 12.3% were nonsense mutations. CONCLUSIONS: Precise genetic characterization in patients with DMD/BMD is timely and important for implementing nationwide registration systems and future molecular therapeutic trials in Korea and globally. Muscle Nerve 55: 727-734, 2017.

GM3 synthase deficiency due to ST3GAL5 variants in two Korean female siblings: Masquerading as Rett syndrome-like phenotype.

There have been a few reports of GM3 synthase deficiency since the disease of the ganglioside biosynthetic pathway was first reported in 2004. It is characterized by infantile-onset epilepsy with severe intellectual disability, blindness, cutaneous dyspigmentation, and choreoathetosis. Here we report the cases of two Korean female siblings with ST3GAL5 variants, who presented with a Rett-like phenotype. They had delayed speech, hand stereotypies with a loss of purposeful hand movements, and choreoathetosis, but no clinical seizures. One of them had microcephaly, while the other had small head circumference less than 10th centile. There were no abnormal laboratory findings with the exception of a high lactate level. MECP2/CDKL5/FOXG1 genetic tests with an array comparative genomic hybridization revealed no molecular defects. Through whole-exome sequencing of the proband, we found compound heterozygous ST3GAL5 variants (p.Gly201Arg and p.Cys195Ser), both of which were novel. The siblings were the same compound heterozygotes and their unaffected parents were heterozygous carriers of each variant. Liquid chromatography-mass spectrometry analysis confirmed a low level of GM3 and its downstream metabolites, indicating GM3 synthase deficiency. These cases expanded the clinical and genetic spectrum of the ultra-rare disease, GM3 synthase deficiency with ST3GAL5 variants. © 2016 Wiley Periodicals, Inc.

Atypical presentation of infantile-onset farber disease with novel ASAH1 mutations.

Farber disease is a very rare autosomal recessive disease caused by mutation of ASAH1 that results in the accumulation of ceramide in various tissues. Clinical symptoms of classic Farber disease comprise painful joint deformity, hoarseness of voice, and subcutaneous nodules. Here, we describe a patient with Farber disease with atypical presentation of early onset hypotonia, sacral mass, congenital heart disease, and dysmorphic face since birth. Severe cognitive disability, failure to gain motor skills, failure to thrive, and joint contractures developed. Using whole-exome sequencing, we identified the compound heterozygote missense mutations of ASAH1 (p.R333C and p.G235R). Because of the diagnostic delay, she underwent sacral mass excision, which revealed enlarged lysosomes and zebra bodies. We report an atypical presentation of Farber disease with her pathology and associated genetic defect. This case expands the phenotypic spectrum of Farber disease to include novel mutations of ASAH1, which pose a diagnostic challenge. We also discuss the clinical utility of whole-exome sequencing for diagnosis of ultra-rare diseases. © 2016 Wiley Periodicals, Inc.

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells.

Pancreatic beta cells have well-developed ER to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by 1D SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. GO analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD001081 (http://proteomecentral.proteomexchange.org/dataset/PXD001081).

Mutational analysis of paediatric patients with tuberous sclerosis complex in Korea: genotype and epilepsy.

To date, only a few studies have reported that, in tuberous sclerosis, TSC2 mutations are more frequently associated with infantile spasms and cognitive impairment compared to TSC1 mutations. We analyzed the mutational spectrum of patients with tuberous sclerosis in Korea and attempted to explore the associations between genotype and seizure type/outcome. We performed mutational analyses on 70 unrelated patients with clinically confirmed tuberous sclerosis by using direct DNA sequencing and/or multiplex ligation-dependent probe amplification. The patients' medical records, including epilepsy type and outcome, were reviewed retrospectively. We identified pathogenic mutations in 55 patients (79%), 25 of which were novel. There were 12 TSC1 mutations and 43 TSC2 mutations. TSC1 mutations included 8 frameshift and 4 nonsense mutations. TSC2 mutations included 12 frameshift, 10 nonsense, 6 splicing, and 6 missense mutations, as well as 4 in-frame deletions and 5 large deletions. Fifty-eight patients had epilepsy (83%), including 19 patients with a history of infantile spasms. Compared to patients with TSC1 mutations, individuals with TSC2 mutations had a significantly higher frequency of epilepsy (p<0.05) and tended to have a higher frequency of infantile spasms (37% vs 17%; p<0.3). Most of the patients with TSC2 mutations who developed infantile spasms exhibited subsequent epilepsy (13/14; 93%). However, the presence/absence of infantile spasms did not influence seizure remission or cognitive outcome.

Rare coincidence of familial central core disease and hemophagocytic lymphohistiocytosis.

Central core disease is a congenital myopathy caused by mutations in RYR1. A 6-year-old girl was admitted due to difficulty in running and climbing stairs. Another 13 members through the four generations had similar symptoms, indicating autosomal dominant inheritance. Muscle biopsy showed the characteristic central cores in predominant type 1 fibers. She later developed hemophagocytic lymphohistiocytosis. Mutation analysis identified c.14582G>A in RYR1, and c.1693delG and c.2954 + 5G>A in UNC13D. To our knowledge, this is the first case of a patient with central core disease, carrying a RYR1 mutation in a Korean large family, who had concurrent familial hemophagocytic lymphohistiocytosis.

Down-regulation of microglial activity attenuates axotomized nigral dopaminergic neuronal cell loss.

BACKGROUND: There is growing evidence that inflammatory processes of activated microglia could play an important role in the progression of nerve cell damage in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease which harbor features of chronic microglial activation, though the precise mechanism is unknown. In this study, we presented in vivo and ex vivo experimental evidences indicating that activated microglia could exacerbate the survival of axotomized dopaminergic neurons and that appropriate inactivation of microglia could be neuroprotective. RESULTS: The transection of medial forebrain bundle (MFB) of a rat induced loss of dopaminergic neurons in a time-dependent manner and accompanied with microglial activation. Along with microglial activation, production of reactive oxygen species (ROS) was upregulated and TH/OX6/hydroethidine triple-immunofluorescence showed that the microglia mainly produced ROS. When the activated microglial cells that were isolated from the substantia nigra of the MFB axotomized animal, were transplanted into the substantia nigra of which MFB had been transected at 7 days ago, the survival rate of axotomized dopaminergic neurons was significantly reduced as compared with sham control. Meanwhile, when the microglial activation was attenuated by administration of tuftsin fragment 1-3 (microglia inhibitory factor) into the lateral ventricle using mini-osmotic pump, the survival rate of axotomized dopaminergic neurons was increased. CONCLUSION: The present study suggests that activated microglia could actively produce and secrete unfavorable toxic substances, such as ROS, which could accelerate dopaminergic neuronal cell loss. So, well-controlled blockade of microglial activation might be neuroprotective in some neuropathological conditions.

Endoplasmic reticulum factor ERLIN2 regulates cytosolic lipid content in cancer cells.

Increased de novo lipogenesis is a hallmark of aggressive cancers. Lipid droplets, the major form of cytosolic lipid storage, have been implicated in cancer cell proliferation and tumorigenesis. Recently, we identified the ERLIN2 [ER (endoplasmic reticulum) lipid raft-associated 2) gene that is amplified and overexpressed in aggressive human breast cancer. Previous studies demonstrated that ERLIN2 plays a supporting oncogenic role by facilitating the transformation of human breast cancer cells. In the present study, we found that ERLIN2 supports cancer cell growth by regulating cytosolic lipid droplet production. ERLIN2 is preferably expressed in human breast cancer cells or hepatoma cells and is inducible by insulin signalling or when cells are cultured in lipoprotein-deficient medium. Increased expression of ERLIN2 promotes the accumulation of cytosolic lipid droplets in breast cancer cells or hepatoma cells in response to insulin or overload of unsaturated fatty acids. ERLIN2 regulates activation of SREBP (sterol regulatory element-binding protein) 1c, the key regulator of de novo lipogenesis, in cancer cells. ERLIN2 was found to bind to INSIG1 (insulin-induced gene 1), a key ER membrane protein that blocks SREBP activation. Consistent with the role of ERLIN2 in regulating cytosolic lipid content, down-regulation of ERLIN2 in breast cancer or hepatoma cells led to lower cell proliferation rates. The present study revealed a novel role for ERLIN2 in supporting cancer cell growth by promoting the activation of the key lipogenic regulator SREBP1c and the production of cytosolic lipid droplets. The identification of ERLIN2 as a regulator of cytosolic lipid content in cancer cells has important implications for understanding the molecular basis of tumorigenesis and the treatment of cancer.

Rapid Targeted Sequencing Using Dried Blood Spot Samples for Patients With Suspected Actionable Genetic Diseases.

Background: New genome sequencing technologies with enhanced diagnostic efficiency have emerged. Rapid and timely diagnosis of treatable rare genetic diseases can alter their medical management and clinical course. However, multiple factors, including ethical issues, must be considered. We designed a targeted sequencing platform to avoid ethical issues and reduce the turnaround time. Methods: We designed an automated sequencing platform using dried blood spot samples and a NEOseq_ACTION panel comprising 254 genes associated with Mendelian diseases having curable or manageable treatment options. Retrospective validation was performed using data from 24 genetically and biochemically confirmed patients. Prospective validation was performed using data from 111 patients with suspected actionable genetic diseases. Results: In prospective clinical validation, 13.5% patients presented with medically actionable diseases, including short- or medium-chain acyl-CoA dehydrogenase deficiencies (N=6), hyperphenylalaninemia (N=2), mucopolysaccharidosis type IVA (N=1), alpha thalassemia (N=1), 3-methylcrotonyl-CoA carboxylase 2 deficiency (N=1), propionic acidemia (N=1), glycogen storage disease, type IX(a) (N=1), congenital myasthenic syndrome (N=1), and citrullinemia, type II (N=1). Using the automated analytic pipeline, the turnaround time from blood collection to result reporting was <4 days. Conclusions: This pilot study evaluated the possibility of rapid and timely diagnosis of treatable rare genetic diseases using a panel designed by a multidisciplinary team. The automated analytic pipeline maximized the clinical utility of rapid targeted sequencing for medically actionable genes, providing a strategy for appropriate and timely treatment of rare genetic diseases.

Oncogenic signaling pathways and hallmarks of cancer in Korean patients with acral melanoma.

Acral melanoma (AM), a rare subtype of cutaneous melanoma, shows higher incidence in Asians, including Koreans, than in Caucasians. However, the genetic modification associated with AM in Koreans is not well known and has not been comprehensively investigated in terms of oncogenic signaling, and hallmarks of cancer. We performed whole-exome and RNA sequencing for Korean patients with AM and acquired the genetic alterations and gene expression profiles. KIT alterations (previously known to be recurrent alterations in AM) and CDK4/CCND1 copy number amplifications were identified in the patients. Genetic and transcriptomic alterations in patients with AM were functionally converge to the hallmarks of cancer and oncogenic pathways, including 'proliferative signal persistence', 'apoptotic resistance', and 'activation of invasion and metastasis', despite the heterogeneous somatic mutation profiles of Korean patients with AM. This study may provide a molecular understanding for therapeutic strategy for AM.

Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

In cells, N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors called SNAREs are involved in membrane fusion. In neurons, for example, target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the pre-synaptic membrane, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, is part of the conserved protein complex involved in neurotransmission. Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes. In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly. Exposure of cholesterol-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP results in dissociated vesicles. In contrast, exposure of LPC-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP, results in inhibition of t-/v-SNARE disassembly and the consequent accumulation of clustered vesicles. Similarly, exposure of isolated rat brain slices and pancreas to cholesterol or LPC, also demonstrates LPC-induced inhibition of SNARE complex disassembly. Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer. The altered plasma LPC levels observed in various cancers may in part contribute to defects in SNARE assembly-disassembly and membrane fusion, consequently affecting protein maturation and secretion in cancer cells.

Water channels in platelet volume regulation.

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein-mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl(2)-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G(αo), G(αi-1) and G(αi-3) proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein-mediated pathway.

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex.

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.

Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models.

The transmembrane 4 L six family member 5 (TM4SF5) is aberrantly expressed in hepatocellular and colorectal cancers, and has been implicated in tumor progression, suggesting that it could serve as a novel therapeutic target. Previously, we screened a murine antibody phage-display library to generate a novel monoclonal antibody, Ab27, that is specific to the extracellular loop 2 of TM4SF5. In this study, we evaluated the effects of chimeric Ab27 using cancer cells expressing endogenous TM4SF5 or stably overexpressing TM4SF5 in vivo and in vitro. Monotherapy with Ab27 significantly decreased tumor growth in liver and colon cancer xenograft models, including a sorafenib-resistant model, and decreased the phosphorylation of focal adhesion kinase (FAK), p27Kip1, and signal transducer and activator of transcription 3 (STAT3). No general Ab27 toxicity was observed in vivo. Combination treatment with Ab27 and sorafenib or doxorubicin exerted higher antitumor activity than monotherapy. In addition, we humanized the Ab27 sequence by the complementarity-determining region (CDR) grafting method. The humanized antibody Ab27-hz9 had reduced immunogenicity but exhibited target recognition and antitumor activity comparable with those of Ab27. Both Ab27 and Ab27-hz9 efficiently targeted tumor cells expressing TM4SF5 in vivo. These observations strongly support the further development of Ab27-hz9 as a novel therapeutic agent against liver and colorectal cancers.

Systematic analysis of inheritance pattern determination in genes that cause rare neurodevelopmental diseases.

Despite recent advancements in our understanding of genetic etiology and its molecular and physiological consequences, it is not yet clear what genetic features determine the inheritance pattern of a disease. To address this issue, we conducted whole exome sequencing analysis to characterize genetic variants in 1,180 Korean patients with neurological symptoms. The diagnostic yield for definitive pathogenic variant findings was 50.8%, after including 33 cases (5.9%) additionally diagnosed by reanalysis. Of diagnosed patients, 33.4% carried inherited variants. At the genetic level, autosomal recessive-inherited genes were characterized by enrichments in metabolic process, muscle organization and metal ion homeostasis pathways. Transcriptome and interactome profiling analyses revealed less brain-centered expression and fewer protein-protein interactions for recessive genes. The majority of autosomal recessive genes were more tolerant of variation, and functional prediction scores of recessively-inherited variants tended to be lower than those of dominantly-inherited variants. Additionally, we were able to predict the rates of carriers for recessive variants. Our results showed that genes responsible for neurodevelopmental disorders harbor different molecular mechanisms and expression patterns according to their inheritance patterns. Also, calculated frequency rates for recessive variants could be utilized to pre-screen rare neurodevelopmental disorder carriers.

Involvement of ß-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release.

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G(αo) -mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G(o) protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous ß-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using ß-adrenergic receptor antibody, and vesicle swelling experiments using ß-adrenergic agonists and antagonists, demonstrate the presence of functional ß-adrenergic receptors at the SV membrane. Since a recent study shows vH(+) -ATPase to be upstream of AQP-6 in the pathway leading from G(αo) -mediated swelling of SV, participation of an endogenous ß-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.