Extended Data Fig. 3: Labeled PIEZO1 displays normal electrophysiological properties.
From: Direct observation of the conformational states of PIEZO1
a, Left, schematic of whole cell electrophysiology with cell poking. Middle, maximal whole cell currents evoked by mechanical stimulation. Cells were transfected with mPIEZO1-IRES-eGFP (WT, grey circles, −1189 ± 280 pA, n = 9 cells), mPiezo1-TCO*K103-C-mEos3.2-TwinStrepTag (blue circles, −1125 ± 262 pA, n = 11 cells), and mPiezo1-TCO*K103-C-HaloTag-TwinStrepTag (red circles, −1259 ± 986 pA, n = 7 cells). All cells were co-transfected with the tRNA/Synthetase and cultured in the presence of the TCO*K unnatural amino acid. Right, time constant of inactivation for maximal whole cell currents shown in the middle (WT = 17 ± 3 ms, C-mEos3.2 = 14 ± 2 ms, C-HaloTag = 13 ± 2 ms). All values are mean ± s.e.m. b, Representative whole cell currents evoked by mechanical stimulation for TCO*K labeled mPIEZO1 with and without the tRNA/synthetase. c, Maximal whole cell currents evoked by mechanical stimulation from cells labeled with tetrazine-AF647 using the same conditions as in a, for cells co-transfected with the tRNA/Synthetase and cultured in the presence of the TCO*K unnatural amino acid. Labeling does not result in a significant change in maximal whole cell current relative to the WT channel (WT = −727 ± 362 pA, n = 4 cells; C-mEos3.2 = −480 ± 128 pA, n = 6 cells; C-HaloTag = −383 ± 108 pA, n = 11 cells). All values are mean ± s.e.m. d, Yoda1-induced slowing of channel inactivation. The time constant of inactivation was measured before, during, and after bath application of 10 µM Yoda1. Lines are shown connecting measurements from individual cells. e, Quantification of Yoda1-induced slowing of inactivation for each of the three constructs tested. The median fold change in inactivation is shown as a black line (WT = 2.8, n = 3 cells; C-mEos3.2 = 4.0, n = 3 cells; C-HaloTag = 2.9, n = 4 cells).
