Fig. 3.
Expression of STIM1 mutants alters the pharmacology and inactivation properties of ICRAC. (A) Measured at a holding potential of −80 mV, ICRAC failed to inactivate in DVF in Jurkat T cells overexpressing the STIM1-ΔM597 deletion mutant, as distinct from endogenous ICRAC (see Fig. 4A Inset) or ICRAC measured after overexpression of hSTIM1 in RBL cells (Fig. 1G). In contrast, other ICRAC properties were similar to endogenous current: activation with passive Ca2+ store depletion, potentiation by 5 μM 2-APB, and inhibition by 50 μM 2-APB. (B) One minute after exposure to DVF, which followed maximal CRAC activation in 20 mM extracellular Ca2+, ICRAC showed ≈25% inactivation when STIM1-ΔM597 was overexpressed (n = 6), significantly different from the ≈80% inactivation observed in STIM1 WT-overexpressing cells (n = 3; P < 0.05). (C) The I–V curve for ICRAC in DVF was unaltered by overexpression of STIM1-ΔM597 (compared with STIM1 WT; Fig. 1E). (D) Assessment of the single-channel conductance by noise analysis (as in Fig. 1H) at −100 mV during inhibition by 2-APB in A revealed no differences from the single-channel current measured for STIM1 WT (Fig. 1H). (E) hSTIM1 E87A mutant was overexpressed after knockdown of rSTIM1 in RBL cells. Sensitivity of ICRAC to 2-APB was greatly altered: ICRAC was immediately inhibited by 5 μM 2-APB, as opposed to the normal potentiation seen in A. (F) Overexpression of STIM1 E87A did not result in any changes in the I–V curve of ICRAC in DVF as compared to STIM1 WT (see Fig. 1E).