Xenbase Image ID: 178537

Figure 9. Cd2+ block mediated by adjacent Kv2.1 I383C subunits predicts that 2:2R heteromers contribute to whole cell currents for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT but not for Kv2.1:Kv6.4 WT. (A) Channels predicted to form for Kv2.1 I383C (black circle with C) + Kv6.4 WT (left, magenta) or Kv2.1 I383C + Kv6.4 PIPIIV-Kv2.1CT (right, blue) at Kv2.1-biased or Kv6.4-biased expression ratios. Cd2+-coordination sites between adjacent cysteine-containing subunits are marked with green circles; all channels with Cd2+-coordination sites are predicted to be sensitive to Cd2+ block. Cd2+-resistant 2:2R heteromers (magenta box) are predicted to contribute to currents in Kv6.4-PIPIIV-Kv2.1CT-biased mixes. (B) Examples current traces before and after addition of 500 µM Cd2+ for Kv2.1, Kv2.1:Kv6.4 and Kv2.1:Kv6.4-PIPIIV-Kv2.1CT recorded in response to a 500-ms +40 mV step following a prepulse of 4 s to −100 mV. Oocytes were held at −70 mV. Experiments were performed in the presence of 200 µM dithiotheitol (DTT), and a 1:10 ratio was used for coexpression experiments. Scale bars indicate current amplitude and time. (C) Fractional current remaining after 500 µM Cd2+ addition determined by measuring the peak current during the +40 mV test pulse using the protocol described in B. Cd2+ block was significantly greater of both heteromer species compared with Kv2.1 homomers (*, P < 0.05, ANOVA + Tukey post hoc, n = 10 for each, bars show SEM). (D) Examples traces before and after application of 500 µM Cd2+ for Kv2.1 I383C, Kv2.1 I383C:Kv6.4 WT, and Kv2.1 I383C:Kv6.4-PIPIIV-Kv2.1CT. Currents were recorded as in B, and a 1:10 Kv2.1:Kv6.4 species expression ratio was used. (E) 500 µM Cd2+-resistant current fraction (peak current during a 2-s test pulse to +40 mV) is shown for Kv2.1 I383C:Kv6.4 WT (magenta) and Kv2.1 I383C:Kv6.4-PIPIIV-Kv2.1CT (blue) at the indicated cRNA expression ratios. The solid gray and dashed gray lines mark the mean ± SEM of the Cd2+-resistant current fraction for Kv2.1I383C homomers. Cd2+ block was significantly reduced at high expression ratios for Kv6.4-PIPIIV-Kv2.1CT compared with the lowest expression ratio used (*, P < 0.05, ANOVA, n = 8–11 for each ratio, bars show SEM). (F) Kv2.1 I383C homomers (n = 10), Kv2.1 I383C:Kv6.4 M422C heteromers (1:10 ratio, n = 12) and Kv2.1:Kv6.4 M422C-PIPIIV-Kv2.1CT (1:11 ratio, n = 9) heteromers, which are all predicted to have 4 Cd2+-coordination sites are highly sensitive to block by 500 µM Cd2+ (**, P < 0.01, ANOVA; *, P < 0.05, ANOVA, bars show SEM). Image published in: Pisupati A et al. (2018) © 2018 Pisupati et al. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

Return to previous page