Journal:

Article Title: Analysis of the Membrane Topology of the Acid-sensing Ion Channel 2a

doi: 10.1074/jbc.M411849200

Figure Lengend Snippet: The DEG/ENaCs consists of intracellular amino and carboxyl termini with two transmembrane domains connected by a large extracellular loop. a, PredictProtein depicts one structure for ASIC2a that is identical to the DEG/ENaC structures. b, TopPred predicts four possible structure outcomes for ASIC2a, one of which is identical to the DEG/ENaC structures. c and d, ConPred II (c) and HMMTOP (d) depict possible alternative structures for ASIC2a.

Article Snippet: The epitope of the ASIC2a antibody (Alomone) is directed against amino acids 1–20 of the intracellular amino terminus; thus, we compared ASIC2a expression in permeabilized versus non-permeabilized cells.

Techniques:

Journal:

Article Title: Analysis of the Membrane Topology of the Acid-sensing Ion Channel 2a

doi: 10.1074/jbc.M411849200

Figure Lengend Snippet: a, the effect of Endo H and PNGase on ASIC2a receptor protein. HEK293 cells transiently expressing the ASIC2a protein were prepared and treated with either Endo H or PNGase. The proteins were separated by SDS-PAGE, transferred to nylon membrane, and the resultant blot was probed with ASIC2a primary antibody (Alomone). The immunoblot shows that both Endo H and PNGase reduced the molecular mass of ASIC2a by ~5 kDa, implying that two glycosylation sites may exist for ASIC2a. b, ASIC2a contains two N-linked glycosylation sites. Mutations N365S and N392S produced ASIC2a channels with a reduced molecular mass, indicating they are both sites for N-linked glycosylation. A double mutant of N365S/N392S produced a reduced molecular mass equal in size to that of PNGase-treated ASIC2a wild type, indicating that only these two sites are required for full glycosylation of ASIC2a. c, the three remaining predicted N-linked glycosylation sites are not glycosylated. Immunoblot analysis of N22S, N478S, and N487S showed no reduction in molecular mass; thus, these ASIC2a proteins are not glycosylated in HEK293 cells. NT, non-transfected.

Article Snippet: The epitope of the ASIC2a antibody (Alomone) is directed against amino acids 1–20 of the intracellular amino terminus; thus, we compared ASIC2a expression in permeabilized versus non-permeabilized cells.

Techniques: Expressing, SDS Page, Western Blot, Produced, Mutagenesis, Transfection

Journal:

Article Title: Analysis of the Membrane Topology of the Acid-sensing Ion Channel 2a

doi: 10.1074/jbc.M411849200

Figure Lengend Snippet: a, pH has a decreased effect at double mutant N365S/N392S. Wild type ASIC2a and double mutant N365S/N392S receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp analysis (−60mV) was carried out by comparing current with decreasing pH. Trace data shows that an increasing level of acidity is required for double mutant activation as compared with wild type ASIC2a. b, only the double mutant N365S/N392S has altered affinity for increased acidity. Predicted glycosylation site mutants N365S (closed box, dash-dot line) and N392S (closed circle, dotted line) are similar to wild type ASIC2a (open box, solid bold line), exhibiting increased inward currents with increasing pH. N365S/N392S (closed diamond, solid line) shows a significant difference (pH50, 2.90 ± 0.14) compared with wild type (pH50, 3.58 ± 0.07).

Article Snippet: The epitope of the ASIC2a antibody (Alomone) is directed against amino acids 1–20 of the intracellular amino terminus; thus, we compared ASIC2a expression in permeabilized versus non-permeabilized cells.

Techniques: Mutagenesis, Activation Assay

Journal:

Article Title: Analysis of the Membrane Topology of the Acid-sensing Ion Channel 2a

doi: 10.1074/jbc.M411849200

Figure Lengend Snippet: The model depicts the membrane topology of ASIC2a based on glycosylation and antibody permeabilization studies. The ASIC2a consists of two TM domains (TM1 and TM2, with amino acid numbers corresponding to the boundaries of the TM domains), intracellular amino and carboxyl termini, and a large extracellular loop. Consensus sites for N-linked glycosylation are shown in black, mutants to introduce consensus N-linked glycosylation sites are shown in gray, and glycosylated residues (H72N, A81N, N365, N392, Y414N, and Y423N/V425S) are shown with the addition of a sugar group.

Article Snippet: The epitope of the ASIC2a antibody (Alomone) is directed against amino acids 1–20 of the intracellular amino terminus; thus, we compared ASIC2a expression in permeabilized versus non-permeabilized cells.

Techniques: Introduce

Journal:

Article Title: Analysis of the Membrane Topology of the Acid-sensing Ion Channel 2a

doi: 10.1074/jbc.M411849200

Figure Lengend Snippet: The anti-ASIC2a epitope is directed against amino acids 1–20. a, ASIC2a-expressing HEK293 cells permeabilized with 3% Triton X-100 are positive for ASIC2a and tubulin. b, ASIC2a-expressing HEK293 cells that are not permeabilized with Triton X-100 show a low level of ASIC2a staining that overlaps with tubulin; thus, these cells are compromised and are permeable to the antibodies. c, wild type HEK293 cells permeabilized with 3% Triton X-100 show no ASIC2a staining but are positive for tubulin. d, ASIC2a-expressing HEK293 cells permeabilized with 3% Triton X-100 but not exposed to the ASIC2a or tubulin antibodies show no ASIC2a or tubulin staining. FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; Ab, antibody.

Article Snippet: The epitope of the ASIC2a antibody (Alomone) is directed against amino acids 1–20 of the intracellular amino terminus; thus, we compared ASIC2a expression in permeabilized versus non-permeabilized cells.

Techniques: Expressing, Staining