Abstract
Acid-sensing ion channels (ASICs) are proton-activated cation channels that play important roles as typical proton sensors during pathophysiological conditions and normal synaptic activities. Among the ASIC subunits, ASIC2a and ASIC2b are alternative splicing products from the same gene, ACCN1. It has been shown that ASIC2 isoforms have differential subcellular distribution: ASIC2a targets the cell surface by itself, while ASIC2b resides in the ER. However, the underlying mechanism for this differential subcellular localization remained to be further elucidated. By constructing ASIC2 chimeras, we found that the first transmembrane (TM1) domain and the proximal post-TM1 domain (17 amino acids) of ASIC2a are critical for membrane targeting of the proteins. We also observed that replacement of corresponding residues in ASIC2b by those of ASIC2a conferred proton-sensitivity as well as surface expression to ASIC2b. We finally confirmed that ASIC2b is delivered to the cell surface from the ER by forming heteromers with ASIC2a, and that the N-terminal region of ASIC2a is additionally required for the ASIC2a-dependent membrane targeting of ASIC2b. Together, our study supports an important role of ASIC2a in membrane targeting of ASIC2b.
Highlights
(CHO) cells[19,20]
ASIC2a was predominantly localized in the cell surface, as evidenced by a yellow color in the merged image of ASIC2a and the plasma membrane marker, Lyn (N-terminal myristoylation and palmitoylation signal sequence from Lyn kinase) (Fig. 1a)
Consistent with our observation in HEK293T cells, ASIC2a was predominantly localized in the cell surface like RFP-PH, as evidenced by overlapped line scanning results of GFP and RFP, while ASIC2b was accumulated in the ER in SH-SY5Y cells (Fig. 7a)
Summary
(CHO) cells[19,20]. surface expression of these mutated channels as well as ASIC2b needs to be further studied. From the perspective of trafficking mechanisms, surface expression of ion channels is largely dependent on discrete motifs that reside in proteins, such as ER retention or export signals. Synthesized proteins containing arginine-based ER retention signals (RXR) or physiologically misfolded and improperly assembled proteins are retained in the ER via the quality control mechanism[29,30,31,32,33,34]. In these cases, ER retention can be antagonized by proper heteromultimeric assembly. By constructing a series of ASIC2 chimeras, we found that the TM1 and the proximal post-TM1 domain of ASIC2a are critical for surface trafficking of ASIC2 channels. Our data show that ASIC2b can be delivered to the cell surface from the ER by heteromeric assembly with ASIC2a, and that the N-terminal region of ASIC2a is necessary for the ASIC2a-dependent membrane targeting of ASIC2b
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