Figure 4. I_Ca,Cl as marker for Ca²⁺ _cyt levels.

I_Ca,Cl was recorded from control cells (untreated), cells treated with thapsigargin (2 μM, 3 h), or injected with 500 μM BAPTA to estimate store Ca²⁺ load and the levels of Ca²⁺ influx. I_Ca,Cl provide endogenous reporters of Ca²⁺ release from stores (I_Cl1) and Ca²⁺ influx from the extracellular space (I_ClT) as described in the text. (A) Voltage protocol and representative current traces of the I_ca,Cl. I_Cl1 is a sustained current recorded upon depolarization to +40 mV (trace t), whereas I_ClT is a transient current detected only when the +40 mV pulse is preceded by a hyperpolarization step to −140 mV (traces x–z). Note that at high 5 mM Ca²⁺ levels, Ca²⁺ influx at −140 mV activates an inward Cl₋ current (trace z). The current traces shown are from the control oocyte in B. The time at which each trace was obtained is indicated in B. (B–G) Oocytes were incubated in Ca²⁺-free Ringer (F-Ca) and treated with ionomycin to release store Ca²⁺. The levels of I_Cl1 induced in response to ionomycin provide a measure of store Ca²⁺ load. I_Cl1 (squares) is plotted as the maximal current at the end of the +40 mV pulse as indicated by the arrow in A (left). After store depletion oocytes were sequentially exposed to solutions containing the indicated Ca²⁺ concentration: L (50 μM Ca²⁺), N (0.6 mM Ca²⁺), H1.5 (1.5 mM Ca²⁺), H3 (3 mM Ca²⁺), and H5 (5 mM Ca²⁺). Store depletion activates Ca²⁺ influx through the SOCE pathway, which activates I_ClT. I_ClT (circles) is plotted as the maximal current during the second +40 mV pulse as indicated by the arrow in A (right). B, D, and F show the time course of I_Cl1 and I_ClT in control, thapsigargin, and BAPTA-treated cells, respectively. The time of solution changes and ionomycin (Ion.) addition are indicated above each panel. C, E, and G show statistical analysis of I_Cl1 and I_ClT. I_Cl1 levels were significantly different in the three treatments (P ≤ 0.0041, n = 5–7). No I_Cl1 was detected in the thapsigargin treatment indicating complete Ca²⁺ store depletion. For I_ClT in each panel the asterisks indicate significantly different means: (C) Con, P ≤ 0.015, n = 5; (E) Thaps, P ≤ 0.00012, n = 7; (G) BAPTA, P = 0.00022, n = 6.