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. 2018 Apr 19;293(22):8626–8637. doi: 10.1074/jbc.RA117.000860

Figure 6.

Figure 6.

N-terminal barttin mutations modify the slow gate open probability of V166E rClC-K1/barttin channels. A, voltage protocol and representative whole-cell current recordings from HEK293T cells expressing rat V166E rClC-K1 channels in the absence or presence of WT barttin. B and C, voltage dependence of relative open probabilities of the fast gates (white symbols), the slow gate (gray symbols), and the total channel (black symbols) in the absence of barttin (B, n = 20) and in the presence of WT barttin (C, n = 13). WT barttin constitutively opens the slow gate of V166E rClC-K1 channels. D, representative whole-cell current recordings from HEK293T cells expressing V166E rClC-K1 channels in the presence of mutant barttin (same voltage protocol as in A). E, voltage dependence of relative slow gate open probabilities of V166E rClC-K1 channels in the presence of mutant barttin. Activation curves of V166E rClC-K1 with WT barttin and without barttin are shown as gray lines. Experiments were conducted under standard conditions using the pRcCMV vector for V166E rClC-K1 protein expression. F and G, representative current recordings and slow gate activation curves for the same mutants as in D and E but using a low expression pSVL vector for V166E rClC-K1 channel proteins, resulting in extensive excess of barttin over ClC-K subunits. H, comparison of relative slow gate open probabilities at +105 mV for standard (black) and low expression (gray) of V166E rClC-K1 channels in the presence of WT or mutant barttin. Broken lines, relative open probabilities of the slow gate at +105 mV for WT barttin (top line, reference condition) or in the absence of barttin (bottom line). Data volume for standard expression/low expression: WT, n = 13/0; Δ2, n = 5/0; Δ2–3, n = 11/6; Δ2–4, n = 8/7; Δ2–5, n = 6/0; Δ2–6, n = 7/4; Δ2–7, n = 6/7; Δ2–8, n = 7/8; Δ5–7, n = 10/5; Δ5–8, n = 4/5; Δ7–8, n = 4/6; Δ8, n = 4/4; Δ1–25, n = 8/7; no barttin, n = 20/0; Δ2–7ins2A, n = 3/8; Δ2–7ins4A, n = 4/6; Δ2–7ins6A, n = 4/12; A2V, n = 8/6; D3N, n = 5/0; D3A, n = 6/7; E4Q, n = 7/6; E4R, n = 5/0; K5A, n = 6/10; K5E, n = 3/8; T6V, n = 10/6; F7A, n = 3/6. Statistical significance was tested by one-way ANOVA and Holm–Sidak post hoc test versus WT barttin: F(45, 265) = 28.7 and p < 0.001. Error bars, S.E. (in B, C, E, and G); S.D. (in H).