Molecular mechanisms underlying surface trafficking and lipid regulation of acid-sensing ion channels (ASICs)

Abstract

Acid-sensing ion channels (ASICs) are proton-activated cation channels that play important roles as typical proton sensors in the nervous system. Protons are released in pain-generating pathophysiological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons also act as a neurotransmitter. Perception of physiological pH changes through ASICs are implicated in nociception, itch, mechanosensation, taste transduction, learning and memory, and fear. In spite of their importance in proton sensing, regulatory mechanisms of these channels still need to be further investigated. In this study, we studied the regulatory mechanisms of ASICs by dividing into two parts. In the first part, we examined whether these channels are regulated by membrane phospholipids, which are general cofactors of many receptors and ion channels. In the second part, we investigated differential surface trafficking mechanisms of ASIC subunits. Firstly, we studied the sensitivity toward membrane phospholipids of ASICs by comparing with that of another proton-sensitive ion channel, transient receptor potential vanilloid 1 (TRPV1) channel. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P2. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P3 in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. Taken together, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P2, and AA, although they have common roles as proton sensors. In the second part, we investigated the regulatory mechanisms of ASICs by revealing surface trafficking mechanisms of ASICs. It is important that newly synthesized receptors or ion channels correctly target their final cellular destinations for their function. Diverse physiological disorders are linked with defects in ion channel surface trafficking. In this study, we focused on ASIC2 subunits in particular. 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. In addition, we also found that ASIC2a has an important role in facilitating ASIC3 surface expression. ASIC2a also efficiently delivered ASIC3 which are predominantly accumulated in the ER with partial distribution in the plasma membrane to the cell surface. We also observed that the ASIC2a-dependent surface trafficking of ASIC3 remarkably enhanced the sustained component of the currents. Our study demonstrates that ASIC2a can increase the membrane conductance sensitivity to protons by facilitating the surface expression of ASIC3 through heteromeric assembly ⓒ 2017 DGIST