Title : Analysis of the membrane topology of the
acid-sensing ion channel 2a
Abstract :
- 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 ap position
- 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