Figure 4: Cytosolic calcium modulated hTPC1 activity.

(a) Recordings of hTPC1-mediated currents at different cytosolic Ca²⁺ concentrations. PI(3,5)P₂ was present in the cytosolic bath solution at 90 nM. Voltages of the main pulse indicated in the left panel. Holding voltage: −70 mV, tail voltage: −50 mV. Standard (luminal) pipette solution with 1 mM CaCl₂ added. (b) Normalized conductances (G_norm) obtained from tail current peaks at −50 mV plotted as a function of the main voltage pulse at different cytosolic Ca²⁺ concentrations. Data from single vacuoles were fitted by Boltzmann function and normalized to the maximum value. Normalised conductances were the average of 4 different vacuoles. Continuous lines represented the Boltzmann fit. (c) Half voltage of activation of the Boltzmann fit shown in B versus cytosolic calcium concentration. (d) Slope of the Boltzmann fit shown in B versus cytosolic calcium concentration. Continuous lines in c and d were obtained by the scheme of (f) (see text and Supplemental Appendix). Data in c and d are shown as mean ± standard deviation. (e) Relaxation time constants versus applied voltage, at different cytosolic calcium concentration. Data from 4 different vacuoles. (f) Allosteric mathematical model describing modulation of hTPC1 by cytosolic Ca²⁺. (g) Same currents, as in A, adding missing traces elicited from −80 up to +90 mV. (h) IV relationships of stationary hTPC1 currents, recorded at different Ca²⁺ concentration. Data were normalised to the current at +50 mV, in 200 μM cytosolic calcium. The continuous lines represented the fitting of the data with the following equation: I = N P_O(V, [Ca²⁺]_cyt) V/(1 + ([Ca²⁺]_cyt/K_OP) exp(2δFV/RT)). The normalisation factor N, the electrical distance δ and the apparent affinity constant for cytosolic calcium K_OP were the free parameters, since the open probability PO was completely determined by the values shown in Supplemental Appendix. A Global fit of the data was not satisfactory, see Supplemental Fig. 2c. Therefore, each data at different cytosolic calcium concentration was fitted separately giving at [Ca²⁺]_cyt = 20 μM: δ = 0.34 and K_OP = 270 μM; at [Ca²⁺]_cyt = 200 μM: δ = 0.44 and K_OP = 610 μM. In the text the average of the two values was reported. The normalisation factor was N = 60.9.