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. 2020 Mar 13;152(5):e201912491. doi: 10.1085/jgp.201912491

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© 2020 Kawanabe et al.

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Figure 1. — Voltage-dependent phosphatase activity of WT and hydrophobic spine mutants of Dr-VSP as estimated from Kir2.1 currents. (A) Schematic view of the protocol used to estimate VSP enzyme activity in the cell-attached patch configuration. The pulse protocol (bottom panel) consisted of a 20-ms step pulse to −100 mV (test pulse to measure Kir2.1 current), followed by a 300-ms depolarization pulse to 100 mV (to induce VSP enzyme activity). This protocol was repeated 21 times without an interval. The holding potential was −30 mV. (B) Representative Kir2.1 current traces recorded from HEK293T cells coexpressing Dr-VSP WT, L223F, or L223Q. All current traces recorded during the 21 repeated stimuli are superimposed during the first 100 ms of the pulse protocol. Inward Kir currents were elicited in hyperpolarizing test pulses, and the current amplitude gradually decreased in accordance with VSP-induced decreases in PI(4,5)P₂ within the plasma membrane. (C) Plots of the time-dependent changes in the normalized amplitudes of the inward Kir currents. Values were measured at the end of the 20-ms hyperpolarizing test pulses (arrows in B) and were normalized by the current amplitude during the first stimulus. Average values were connected by lines. Gray unfilled circles (WT), black squares (L223F) and black unfilled triangles (L223Q) were drawn based on data shown in B. Symbols depict the mean ± SD of data from 17 WT, 22 L223F, or 4 L223Q cells. (D) Phosphatase activities of Dr-VSP WT and hydrophobic spine mutants (WT, L223F, L223Q, and Y224Q) estimated as rate constants for the decrease in normalized Kir currents with single exponential fitting. Fitting was applied to the dataset from each cell, then rate constants were averaged. Data are the mean ± SD from 3 to 22 cells, and each symbol shows the data from each cell. Values in parentheses indicate the numbers of cells. Upper bars show P values from a two-tailed Student’s t test.