Extended Data Fig. 2: Whole-cell recordings of CBTA-binding site mutants of TRPM5.
From: A single allosteric site merges activation, modulation and inhibition in TRPM5
a, Representative whole-cell current traces recorded using a ramp protocol from −100 to +100 mV in non-transfected tsA201 cells, cells transfected with wild-type TRPM5 and its mutants F730A, L764A, R834A, R998A, N723A. Black and red traces show baseline currents and currents with 20 μM CBTA, respectively. Pipette solution contains 5 mM EGTA. b, Estimated marginal means (EMMs) of CBTA-activated currents after adjusting for 1 µM free Ca2+-evoked currents using ANCOVA. c, Normalized adjusted CBTA responses. For both b and c, WT n = 5 and mutants n = 4 each. Circles represent mean, and error bars represent s.e.m. Method: Whole-cell currents evoked by 1 µM free Ca2+ served as a proxy for channel expression. Because Ca2+- and CBTA-activated currents were obtained from different cells, genotype-level median Ca2+ currents were assigned as expression values. Both CBTA and Ca2+ currents were log-transformed. An ANCOVA model (Python, statsmodels) was fitted with log(I_CBTA) as the dependent variable, log(I_Ca) as the covariate, and genotype as the fixed factor (WT as the reference). EMMs were extracted at the average Ca2+ expression level. d, Representative plot showing currents measured at +100 and −100 mV, with time 0 indicating immediately after whole-cell configuration. The pipette solution contains 1 μM free calcium. Peak and steady-state refer to the largest current and the stabilized current observed over 30 s, respectively. e, Peak currents measured at +100 and −100 mV for non-transfected tsA201 (n = 6), WT TRPM5 (n = 9), and mutants (F730A, n = 10; L764A, n = 10; R834A, n = 12; R998A, n = 10; N723A, n = 10). Bars represent mean; each dot corresponds to an independent cell measurement. f, Steady-state currents measured at +100 and −100 mV for non-transfected tsA201 cells (n = 6), WT TRPM5 (n = 9) and mutants (F730A, n = 10; L764A, n = 10; R834A, n = 12; R998A, n = 10; N723A, n = 10). Bars represent mean; each dot corresponds to an independent cell measurement. g, Representative traces of peak and steady-state currents recorded from non-transfected tsA201 cells, WT TRPM5 and mutants using a ramp protocol from −100 to +100 mV. The pipette solution contains 1 μM free calcium.
