(A) Macroscopic inward T5L-TRPM2 current in inside-out patch excised from Xenopus laevis oocyte, elicited at -20 mV by repeated exposures to saturating Ca2+ (black bars) and 1 or 100 μM ADPR (blue bars), in the absence or presence of 100 μM ε-ADPR (red bars). (B) Dose-dependence of TRPM2 activation by ADPR in the absence (blue symbols and fit line) and presence (violet symbols and fit line) of 100 μM ε-ADPR (mean ± SEM, n≥7). Printed parameters KADPR, n, and Kε-ADPR reflect the apparent affinity and Hill slope for ADPR binding, and KI for ε-ADPR binding, respectively, obtained from the fits (see Materials and methods). (C) Fluorescence intensity (arbitrary units, Ex: 310 nm, Em: 420 nm) of ε-ADPR at various total concentrations in the absence of protein (black symbols), or in the presence of 100 μM of either BSA (gray symbols) or Chi4S (red symbols). Symbols plot mean ± SD from four, two, and two independent titrations, respectively, for buffer (black), BSA (gray), and Chi4S (red). Black line is a smooth fit of the black symbols by a three-parameter empirical mathematical function, and was used as a calibration curve to determine free [ε-ADPR] in the presence of Chi4S, assuming negligible fluorescence of the bound analog (red projection lines). (D) Plot of upper estimate of free [ε-ADPR] as a function of total [ε-ADPR] in the presence of 100 μM Chi4S (red symbols), fitted (red line) to a simple binding equation (see Materials and methods) to obtain an upper estimate of Kd (see panel). Black line illustrates free [ε-ADPR] expected in the absence of binding.