Molecular Mechanism of Constitutive Endocytosis of Acid-Sensing Ion Channel 1a and Its Protective Function in Acidosis-Induced Neuronal Death

Abstract

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the peripheral and CNSs, which critically contribute to a variety of pathophysiological conditions that involve tissue acidosis, such as ischemic stroke and epileptic seizures. However, the trafficking mechanisms of ASICs and the related proteins remain largely unknown. Here, we demonstrate that ASIC1a, the main ASIC subunit in the brain, undergoes constitutive endocytosis in a clathrin- and dynamin-dependent manner in both mouse cortical neurons and heterologous cell cultures. The endocytosis of ASIC1a was inhibited by either the small molecular inhibitor tyrphostin A23 or knockdown of the core subunit of adaptor protein 2 (AP2) μ2 using RNA interference, supporting a clathrin-dependent endocytosis of ASIC1a. In addition, the internalization of ASIC1a was blocked by dominant-negative dynamin1 mutation K44A and the small molecular inhibitor dynasore, suggesting that it is also dynamin-dependent. We show that the membrane-proximal residues 465LCRRG 469 at the cytoplasmic C terminus of ASIC1a are critical for interaction with the endogenous adaptor protein complex and inhibition of ASIC1a internalization strongly exacerbated acidosis-induced death of cortical neurons from wild-type but not ASIC1a knock-out mice. Together, these results reveal the molecular mechanism of ASIC1a internalization and suggest the importance of endocytic pathway in functional regulation of ASIC1a channels as well as neuronal damages mediated by these channels during neurodegeneration.

Three distinct motifs within the C-terminus of acid-sensing ion channel 1a regulate its surface trafficking

“…Here, we performed a thorough examination of ASIC1a C-terminal tail and revealed several distinct motifs regulating ASIC1a surface expression and channel properties. Of note, the RRGK motif overlaps with a recently reported LCRRG motif (Zeng et al, 2013, see below for more discussion). These data provide a foundation for future studies of the molecular mechanisms that regulate ASIC1a trafficking and function.…”

“…In a recent study, an LCRRG motif, which overlaps with the RRGK motif reported here, is required for the interaction between ASIC1a and AP2, which induces endocytosis of ASIC1a (Zeng et al, 2013). However, the authors also commented that they were unable to assess the endocytosis of the LCRRG mutants using surface biotinylation analysis, because their unpublished data suggest that the same mutants also abolished ASIC1a forward trafficking.…”

“…We and others have recently revealed the importance of ASIC1a trafficking in acidosis-induced changes in neurons (Chai et al, 2007, Chai et al, 2010, Jin et al, 2010, Jing et al, 2011, Duan et al, 2012, Jing et al, 2012, Zeng et al, 2013). However, we remain know little about the molecular mechanisms regulating ASIC trafficking.…”

Acid-sensing ion channels in pain and disease

“…Building on these observations, a recent paper observed that constitutive endocytosis of ASIC1A in a clathrin- and dynamin-dependent manner protected cells against acid-induced cell death 77 . Potential roles of the other ASICs, particularly ASIC2B, have been uncertain, as ASIC2B homomers do not form pH-sensitive channels 7,27 .…”

Neurorestoration after stroke

“…An acid-sensing ion channel, ASIC1a, is involved in the Ca 2+ influx-inhibiting this channel may be neuro-protective. 154 In addition to the Ca 2+ influx, failure of Ca 2+ efflux contributes to the Ca 2+ accumulation. Prostaglandin E2 EP1 receptors have been implicated in the failure of the Na + /Ca 2+ exchanger during ischemia.…”