Analysis of the membrane topology of the acid-sensing ion channel 2a.
Acid-sensing ion channels, or ASICs, are members of the amiloride-sensitive cationic channel superfamily that are predicted to have intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop. This prediction comes from biochemical studies of the mammalian epithelial sodium channels where glycosylation mutants identified the extracellular regions of the channel and a combination of antibody sensitivity and protease action substantiated the intracellular nature of the amino and carboxyl termini. However, although there are highly conserved regions within the different cation channel family members, membrane topology prediction programs provide several alternative structures for the ASICs. Thus, we used glycosylation studies to define the actual membrane topology of the ASIC2a subtype. We deleted the five predicted endogenous asparagine-linked glycosylation sites (Asn-Xaa-(Ser/Thr)) at Asn-22, Asn-365, Asn-392, Asn-478, and Asn-487 to map the extracellular topology. We then introduced exogenous asparagine-linked glycosylation sites at Lys-4, Pro-37, Arg-63, Tyr-67, His-72, Ala-81, Tyr-414, Tyr-423, and Tyr-453 to define the transmembrane domain borders. Finally, we used cell permeabilization studies to confirm the intracellular amino termini of ASIC2a. The data show that Asn-365 and Asn-392 are extracellular and that the introduction of asparagine-linked glycosylation sites at His-72, Ala-81, Tyr-414, and Tyr-423 leads to an increase in molecular mass consistent with an extracellular apposition. In addition, heterologous expression of ASIC2a requires membrane permeabilization for antibody staining. These data confirm the membrane topology prediction that the ASIC2a subtype consists of intracellular amino and carboxyl termini and two transmembrane domains connected by a large extracellular loop.
Saugstad
Julie A
JA
Robert S. Dow Neurobiology Laboratories, Legacy Research, 1225 N.E. 2nd Avenue, Portland, OR 97232, USA. JSaugstad@DowNeurobiology.org
Roberts
Jonathan A
JA
Dong
Jin
J
Zeitouni
Suzanne
S
Evans
Richard J
RJ
D062565:Acid Sensing Ion Channels; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D007477:Ions; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection
D062565:Acid Sensing Ion Channels; D018076:DNA, Complementary; D007477:Ions; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015222:Sodium Channels