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Dale J. Benos - One of the best experts on this subject based on the ideXlab platform.

  • two pkc consensus sites on human acid sensing ion channel 1b differentially regulate its function
    American Journal of Physiology-cell Physiology, 2009
    Co-Authors: Edlira Bashari, Yawar J. Qadri, Catherine M. Fuller, Zhenhong Zhou, Niren Kapoor, Susan J Anderson, Robert H Meltzer, Dale J. Benos
    Abstract:

    Human acid-sensing ion channel 1b (hASIC1b) is a H+-gated Amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an Amiloride-sensitive cation conductance. Amiloride...

  • the binding site on cochlear stereocilia for antisera raised against renal na channels is blocked by Amiloride and dihydrostreptomycin
    Hearing Research, 1996
    Co-Authors: David N Furness, Carole M Hackney, Dale J. Benos
    Abstract:

    Abstract The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be Amiloride-sensitive and immunogold labelling with antibodies raised against the Amiloride-sensitive epithelial Na + channel from kidney (αNaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if Amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with αNaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with Amiloride and then trypsinized in its presence. Because Amiloride is known to protect Amiloride-binding sites from degradation by trypsin, these results suggest that αNaCh is revealing Amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with αNaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na + recognises a site with characteristics similar to the mechanoelectrical transduction channels.

  • Amiloride sensitive channels in marginal cells in the stria vascularis of the guinea pig cochlea
    Neuroscience Letters, 1994
    Co-Authors: Kuni H Iwasa, Dale J. Benos, Kunihiro Mizuta, David J Lim, Masayoshi Tachibana
    Abstract:

    We examined marginal cells in stria vascularis for the presence of Amiloride-sensitive Na+ channels, a possible pathway for maintaining a low Na+ concentration in the endolymph. Whole-cell voltage-clamp experiment shows that Amiloride at 1 microM concentration reversibly reduces inward current more than outward current. Immunogold-labeling method shows that the luminal and lateral membrane have antigenic sites for these antibodies. These observations indicate the presence of Amiloride-sensitive channels in the marginal cell. If Amiloride-sensitive channels in the luminal membrane are highly selective to Na+, they could be an efficient pathway for Na+ uptake from the endolymph. In the basolateral membrane, Amiloride-sensitive Na+ channels may make a relatively small contribution to the unusual resting potential.

  • epithelial sodium conductance in rabbit preimplantation trophectodermal cells
    Developmental Biology, 1991
    Co-Authors: Douglas H Robinson, James K. Bubien, Peter R. Smith, Dale J. Benos
    Abstract:

    Abstract We examined the development of epithelial Na+ conductance in 6- and 7-day post coitus (p.c.) preimplantation rabbit embryos using the whole-cell patch-clamp technique on dissociated rabbit trophectodermal cells and by immunocytochemical localization using a polyclonal antibody directed against subunits of an apical epithelial Na+ channel on the intact blastocyst. In Day 6 and 7 p.c. trophectodermal cells, we observed an outwardly rectified whole-cell Na+ current. The current-voltage characteristics did not differ between the 6- and the 7-day p.c. cells. Replacement of Na+ with the impermeant cation N-methyl- d -glucamine in the pipette or bath reduced outward currents and inward currents, respectively, indicating that the current was Na+-dependent. Treatment of 7-day p.c. cells with 100 μM Amiloride, benzamil, or ethylisopropyl Amiloride (EIPA) blocked the whole-cell currents within 5 min. However, the current of the Day 6 p.c. embryo was not blocked by Amiloride. The Amiloride block at Day 7 p.c. was only partially reversible after 15 min of continuous perfusion of the bath with an Amiloride-free solution. The apparent dissociation constant (Ki) for Amiloride, benzamil, and EIPA was 12, 50, and 16 μM, respectively, when measured 5 min after drug addition. Immunolocalization studies of blastocysts with a polyclonal antibody raised against a high Amiloride affinity Na+ channel isolated from bovine kidney revealed no specific binding to the trophectodermal cells at Day 6 p.c. At Day 7 p.c., however, an epitope, immunoreactive to the high Amiloride affinity Na+ channel antibody, was predominantly localized to the microvilli on the apical membrane of the trophectodermal cells. These findings suggest that: (1) Day 6 and 7 p.c. trophectodermal cells have a Na+ conductance that displays similar current-voltage characteristics; (2) the apical expression of an epitope, possibly a protein related to an epithelial sodium channel, occurs between Days 6 and 7 p.c.; (3) an Amiloride-sensitive component of the sodium current develops between Days 6 and 7 p.c.; and (4) the expression of immunoreactivity and development of Amiloride sensitivity are temporally related.

Jacek Lipkowski - One of the best experts on this subject based on the ideXlab platform.

  • effects of Amiloride an ion channel blocker on alamethicin pore formation in negatively charged gold supported phospholipid bilayers a molecular view
    Langmuir, 2019
    Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek Lipkowski
    Abstract:

    The effects of Amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when Amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that Amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that Amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged Amiloride molecules and the negative charge on the membrane.

  • Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View
    2019
    Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarez-malmagro, Jacek Lipkowski
    Abstract:

    The effects of Amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when Amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that Amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that Amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged Amiloride molecules and the negative charge on the membrane

Fatemeh Abbasi - One of the best experts on this subject based on the ideXlab platform.

  • effects of Amiloride an ion channel blocker on alamethicin pore formation in negatively charged gold supported phospholipid bilayers a molecular view
    Langmuir, 2019
    Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek Lipkowski
    Abstract:

    The effects of Amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when Amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that Amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that Amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged Amiloride molecules and the negative charge on the membrane.

  • Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View
    2019
    Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarez-malmagro, Jacek Lipkowski
    Abstract:

    The effects of Amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when Amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that Amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that Amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged Amiloride molecules and the negative charge on the membrane

Stephen A Ernst - One of the best experts on this subject based on the ideXlab platform.

  • characterization and cellular localization of the epithelial na channel studies using an anti na channel antibody raised by an antiidiotypic route
    Journal of Biological Chemistry, 1991
    Co-Authors: Thomas R Kleyman, Jean Pierre Kraehenbuhl, Stephen A Ernst
    Abstract:

    Abstract Amiloride-sensitive Na+ channels are expressed at the apical membrane of high resistance, Na+-transporting epithelial. The specific interaction of Amiloride with this transport protein suggested the feasibility of raising anti-Na+ channel antibodies by an antiidiotypic approach designed to generate antibodies directed against the Amiloride-binding domain on the channel. Antiidiotypic monoclonal antibody RA6.3 mimicked the effect of Amiloride by inhibiting Na+ transport across A6 cell monolayers when applied to the apical cell surface. Inhibition of transport required pretreatment of the apical cell surface with trypsin in the presence of Amiloride in order to enhance accessibility of the antibody to the Amiloride-binding site. This antibody specifically immunoprecipitated a large 750,000-700,000 Da protein from [35S]methionine-labeled A6 cell cultures, which was resolved further under reducing conditions as a set of polypeptides with apparent molecular masses of 260,000-230,000, 180,000, 140,000-110,000, and 70,000 Da. The antibody recognized the 140,000-Da subunit, known to contain the Amiloride-binding domain, on immunoblots of purified A6 cell Na+ channel. Immunoprecipitation of apical or basolateral plasma membrane proteins selectively labeled with 125I demonstrated that expression of the oligomeric Na+ channel was restricted to the apical plasma membrane. Immunocytochemical localization in A6 cultures revealed apical membrane as well as cytosolic immunoreactive sites. Immunostaining was also observed at or near the basolateral plasma membrane.

Jay J. Leitch - One of the best experts on this subject based on the ideXlab platform.

  • effects of Amiloride an ion channel blocker on alamethicin pore formation in negatively charged gold supported phospholipid bilayers a molecular view
    Langmuir, 2019
    Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek Lipkowski
    Abstract:

    The effects of Amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when Amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that Amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that Amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged Amiloride molecules and the negative charge on the membrane.

  • Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View
    2019
    Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarez-malmagro, Jacek Lipkowski
    Abstract:

    The effects of Amiloride on the structure and conductivity of alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing alamethicin decreases by an order of magnitude when Amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that Amiloride does not inhibit ion channel formation by alamethicin peptides. High-resolution AFM images revealed that Amiloride enlarges and distorts the shape of alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the alamethicin pores. This effect is driven by electrostatic interactions between positively charged Amiloride molecules and the negative charge on the membrane