Figure 2. Incorporation of L-ANAP into ASIC1 produces full-length channel with normal pH-dependent gating.

(A) Representative western blots for rASIC1a expressed in CHO-K1 cells with a single TAG mutation at denoted position, blotted with an anti-GFP antibody. Each TAG mutant was cultured both with L-ANAP (10μM) and without L-ANAP supplementation. Expression of channels with L-ANAP incorporated is seen with bands at ~ 75 kDa. Higher molecular weight bands are likely channel oligomers (dimer, trimer) and bands below are likely free mCitrine. Neither the higher nor lower bands make up a significant portion of channel expressed. Less protein was loaded in the WT (no TAG) control C469 WT. (B) Representative whole-cell recordings of TAG mutants with single cysteine at C469 (colors) and C469 WT construct (black) elicited by solution switch from pH 8 to pH 5.5. (C) pH dependence of activation for rASIC1a TAG mutants with single cysteine at C469 (colors) and the C469 WT construct (black). Data were fit to a modified Hill equation. Fit results found in Figure 2 -table supplement 1.