W287G and Y71G show increased resistance to tryptic cleavage.

<p>CHO cells were transfected with wild-type, W287G or Y71G ASIC1a. Following biotinylation of surface proteins, cells were treated with 0, 1, or 10 µg/ml trypsin for 20 mins. Surface proteins were isolated and blotted for ASIC1a. Note that wild-type ASIC1a at the surface was cleaved by trypsin in a dosage-dependent manner. Also note that the amount of uncleaved mutants was comparable to that of uncleaved WT, although the mutants only had a small fraction of cleaved population (see the bottom image with level enhanced). The decrease in loading in trypsin treated lanes was likely due to the loss of some cells during the treatment. Blots shown are representative from three separate experiments.</p

Lowering the culture temperature increases the current amplitude and slows the rate of desensitization of ASIC1a.

<p>CHO cells were co-transfected with wild-type, W287G or Y71G ASIC1a together with GFP. Cells were cultured overnight at 37°C followed by 24 hrs at 37°C or 27°C. All recordings were performed at the same temperature (∼23°C). (<b>A</b>) Representative pH 6-induced current from cells expressing wild-type or mutant ASIC1a. (<b>B & C</b>) Quantification of the effect of 27°C on current amplitude (B) and the rate of desensitization (C) of wild-type ASIC1a. Asterisks indicate significant differences from 37°C (P<0.01, student's t-test).</p

Mutating Tyr71 and Trp287 reduces ASIC1a surface expression.

<p>(<b>A</b>) The effect of N- and C-terminal tags on surface expression of ASIC1a. CHO cells were transfected with untagged, N-terminal or C-terminal HA- or Flag-tagged ASIC1a as indicated. Surface and total proteins were blotted with a goat anti-ASIC1 and a mouse anti-tubulin antibody, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026909#s4" target="_blank">Methods</a>. Right panel shows the quantification of the surface:total ratio of untagged and tagged ASIC1a. The dotted line indicates that the controls for HA and Flag tagged experiments were different. Average surface:total ratio for WT was 4.4%±0.5%. For easy comparison, the ratio of WT was set arbitrarily to 1. (<b>B</b>) Typical traces and quantification of pH6-activated current recorded from cells expressing untagged (n = 6) or N-terminal HA-tagged (n = 7) ASIC1a. (<b>C</b>) Western blot and quantification showing surface:total ratio of HA-tagged wild-type ASIC1a, W287G, or Y71G mutants. Asterisks indicate a significant difference from controls (P<0.01, unbalanced ANOVA).</p

W287G and Y71G reduces ASIC1a glycosylation while a reduced culture temperature has an opposite effect.

<p>(<b>A & B</b>) Representative blots showing the effect of a reduced culture temperature on ASIC1a glycosylation. CHO cells were transfected with wild-type ASIC1a, W287G or Y71G mutant. Surface biotinylation was performed after culturing at 37°C (A) or 27°C (B) for 24 hrs. Lysates were treated with PNGase F or Endo H as indicated, followed by NeutrAvidin precipitation. Surface and total fraction were blotted for ASIC1a. The relative position of Endo H-resistant and -sensitive ASIC1a was indicated by arrows. Note that 27°C increased the percentage of Endo H-resistant wild-type ASIC1a in total lysate. Also note the appearance of Endo H-resistant W287G and Y71G at the cell surface at 27°C (arrows). (<b>C</b>) Quantification of Endo H-resistant wild-type ASIC1a at 37°C and 27°C. Asterisk indicates significant difference (P = 0.025); n.s. stands for not significant.</p