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. 2023 Jan 20;122(11):2267–2284. doi: 10.1016/j.bpj.2023.01.022

Figure 2.

Figure 2

Characterization of voltage-dependent phosphoinositide phosphatase activity of the sea squirt-opossum VSP chimera, Ci-Md-VSP in Xenopus oocyte. (A) Four subreactions of PIPs dephosphorylation mediated by VSP. (B) Amino acid sequences around the junction region of chimeric construction between Ci-VSP and Md-VSP. Ci-Md, Md, and Ci denote the Ci-Md-VSP, Md-VSP, and Ci-VSP, respectively. (C) A scheme of FRET probe, F-PLC or F-TAPP, for sensing PI(4,5)P2 or PI(3,4)P2, respectively, consisting of the two fluorescent proteins (box) sandwiching the PH domain (gray) selective to each species of PIP. (D) Representative traces for YFP/CFP ratio of F-PLC (left) and F-TAPP (right) with Ci-VSP (black), Ci-Md-VSP (red), or without VSP (no VSP, purple) at indicated voltage. Traces are normalized to baseline level of fluorescence before depolarization. Depolarizing step shown at top of traces was applied from a holding potential of −60 mV to indicated value for 5 s. Dotted line in each trace indicates the time point (at 4 s after the beginning of depolarization) for calculating ΔFRET. (E) Graph showing ΔFRET for F-PLC (top) and F-TAPP (bottom) at six voltages for Ci-Md-VSP and Ci-VSP. The number of recordings is shown within the graph. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s., statistically not significant, two-tailed unpaired t-test. Data are presented as mean ± SE. (F) Left: representative GIRK2 traces in oocytes expressing Ci-Md-VSP. GIRK2 currents were evoked by hyperpolarizing steps from the holding potential of −60 to −120 mV and then oocytes were depolarized to 0, 50, or 100 mV for 300 ms. This was repeated 12 times, and all GIRK2 traces are superimposed (see also Fig. S3). The 12th traces are shown in red. Black dotted lines indicate zero current level. Right: time course of GIRK2 current upon voltage-evoked VSP activities with five levels of conditioning depolarization. The current amplitudes were normalized to that in the first trace. Data are presented as mean ± SE. (G) Decay rate constant of the normalized GIRK2 current (s−1) at different potentials. GIRK2 was coexpressed with Ci-VSP or Ci-Md-VSP. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s., statistically not significant, two-tailed unpaired t-test. Data are presented as mean ± SE.