Abstract
2-Aminoethoxydiphenyl borate (2-APB) elicits potentiation current (Ip) on Ca2+ release-activated Ca2+ (CRAC) channels. An accurate investigation into this modulation mechanism would reveal how STIM1-dependent channel gating is enhanced, and benefit the future immune enhancer development. Here, we directly probed the pore diameter of CRAC channels and found that 2-APB enlarged the pore size of STIM1-activated Orai1 from 3.8 to 4.6 Å. We demonstrated that ions with small sizes, i.e., Ca2+ and Na+, mediated prominent 2-APB-induced Ip on the wildtype (WT) Orai1 channels of narrow pore sizes, while conducted decreased or no Ip on Orai1-V102C/A/G mutant channels with enlarged pore diameters. On the contrary, large Cs+ ions blocked the WT channels, while displayed large 2-APB induced Ip on pore-enlarged Orai1-V102C/A/G mutant channels, and the potentiation ratio was highest on Orai1-V102C with an intermediate pore size. Furthermore, we showed that 2-APB potentiated Cs+ current on constitutively active Orai1-V102C/A/G mutants independent of STIM1. Our data suggest that 2-APB directly dilates the pore of open Orai1 channels, both ion size and pore diameter jointly determine the amplitude of Ip on CRAC channels, and the generation of Ip requires the open state of Orai1, not STIM1 itself.
Highlights
The Ca2+ release-activated Ca2+ (CRAC) channels play an essential and specific role in immune system
A high dose of 2-APB exhibits prominent agonistic activity on Orai[3], and to a much less degree on STIM1-free Orai[1] channels[16,18]. This direct-gating effect of 2-APB on Orai[3] and Orai[1] was characterized by biphasic inwardly and outwardly rectified currents and differed from potentiation effect in many aspects, such as 1) loss of high Ca2+ selectivity and altered I-V profile; 2) increased permeability to Cs+ due to enlarged pore diameter of Orai319,20; 3) STIM1-bound channels, especially Orai[1], resist this 2-APB direct gating effect[21], whereas the generation of Ip is STIM1-activation dependent; 4) different dose requirements: the reported EC50 for 2-APB-induced Ip was 3.1 μMfor native CRAC channels[15] and was 4 μMfor STIM1-activated Orai[1] channels[18], while the reported EC50 for 2-APB-direct gating on Orai[1] alone was 20 ± 1 μM, and that for Orai[3] alone was 14 ± 4 μM18
Since the STIM1-free Orai1-E106D mutant became susceptible to 2-APB direct gating, it was suggested that 2-APB facilitated CRAC channels by altering the pore architecture[18]
Summary
We have investigated the underlying mechanism for 2-APB-elicited Ip by directly probing the pore diameter of CRAC channels and understanding how ion size and channel pore diameter contribute to the generation and amplitude of Ip. Our data strongly suggest that 2-APB facilitates more ions passing through every single channel per unit time via the pore dilation effect, generates larger Ip by increasing i rather than Po or N To directly validate this hypothesis, we performed nonstationary noise analysis of 2-APB effect on native (Jurkat cells) as well as recombinant (Orai[1] and STIM1 transfected HEK293 cells) CRAC channels employed the same protocol as previously reported[20,31]. The resulting 2-APB induced Ip on Orai[1] largely resembles the properties of native ICRAC, preventing aberrant ion signals from interfering with the normal immune reaction Both STIM1-mediated gating can be enhanced and high Ca2+ selectivity of Icrac can be sustained by the pore-dilation effect of 2-APB; as an ideal model for an immune enhancer, the activity-dependent potentiation effect of 2-APB on Orai[1] should be employed in the development of specific immune enhancers. Further work involving structural studies and the site-directed mutagenesis of CRAC channel proteins should help to solve this challenge
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