Dynamic landscape of the intracellular termini of acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are trimeric proton-gated sodium channels. Recent work has shown that these channels play a role in necroptosis following prolonged acidic exposure like occurs in stroke. The C-terminus of ASIC1a is thought to mediate necroptotic cell death through interaction with receptor interacting serine threonine kinase 1 (RIPK1). This interaction is hypothesized to be inhibited at rest via an interaction between the C- and N-termini which blocks the RIPK1 binding site. Here, we use two transition metal ion FRET methods to investigate the conformational dynamics of the termini at neutral and acidic pH. We do not find evidence that the termini are close enough to be bound while the channel is at rest and find that the termini may modestly move closer together during acidification. At rest, the N-terminus adopts a conformation parallel to the membrane about 10 Å away. The distal end of the C-terminus may also spend time close to the membrane at rest. After acidification, the proximal portion of the N-terminus moves marginally closer to the membrane whereas the distal portion of the C-terminus swings away from the membrane. Together these data suggest that a new hypothesis for RIPK1 binding during stroke is needed.

Dynamic landscape of the intracellular termini of acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are trimeric proton-gated sodium channels. Recently it has been shown that these channels play a role in necroptosis following prolonged acidic exposure like occurs in stroke. The C-terminus of the channel is thought to mediate necroptotic cell death through interaction with receptor interacting serine threonine kinase 1 (RIPK1). This interaction is hypothesized to be inhibited at rest via an interaction between the C-terminus and the N-terminus which blocks the RIPK1 binding site. Here, we use a combination of two transition metal ion FRET methods to investigate the conformational dynamics of the termini while the channel is closed and desensitized. We do not find evidence that the termini are close enough to be bound while the channel is at rest and find that the termini may modestly move closer together when desensitized. At rest, the N-terminus adopts a conformation parallel to the membrane about 10 Å away. The distal end of the C-terminus may also spend time close to the membrane at rest. After acidification, the proximal portion of the N-terminus moves marginally closer to the membrane whereas the distal portion of the C-terminus swings away from the membrane. Together these data suggest that a new hypothesis for RIPK1 binding during stroke is needed.

Proliferation in the developing intestine is regulated by the endosomal protein Endotubin.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTBfl/fl mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.