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 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.
Highlights
Introduction of Potential NLinked Glycosylation Sites—To investigate further the membrane topology of ASIC2a, we generated a series of mutant channels to introduce consensus sequences for glycosylation
We generated mutants first to investigate the regions that are predicted to be in the intracellular domains (K4N and P37N of the amino terminus compared with N478S and N487S of the carboxyl terminus) and second, to define the regions post-TM1 (R63N, Y67N, H72N, and A81N) and pre-TM2 (Y414N and Y423N/V425S) that can be glycosylated (Fig. 4)
The post-TM1 mutants H72N and A81N showed an increase in molecular mass compared with WT channels, demonstrating that these residues are glycosylated, whereas R63N and Y67N had no effect on the molecular mass of the Acid-sensing ion channels (ASICs) channel (Fig. 4b)
Summary
Introduction of Potential NLinked Glycosylation Sites—To investigate further the membrane topology of ASIC2a, we generated a series of mutant channels to introduce consensus sequences for glycosylation. The pre-TM2 mutants Y414N and Y423N/V425S both resulted in a doublet of bands corresponding to the WT channel and an additional extra-glycosylated form, whereas the post-TM2 mutant Y453N/Y455S did not result in any change in molecular mass of the ASIC2a protein (Fig. 4c).
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