Abstract
The mechanism for coupling between Ca(2+) stores and store-operated channels (SOCs) is an important but unresolved question. Although SOCs have not been molecularly identified, transient receptor potential (TRP) channels share a number of operational parameters with SOCs. The question of whether activation of SOCs and TRP channels is mediated by the inositol 1,4,5-trisphosphate receptor (InsP(3)R) was examined using the permeant InsP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB) in both mammalian and invertebrate systems. In HEK293 cells stably transfected with human TRPC3 channels, the actions of 2-APB to block carbachol-induced InsP(3)R-mediated store release and carbachol-induced Sr(2+) entry through TRPC3 channels were both reversed at high agonist levels, suggesting InsP(3)Rs mediate TRPC3 activation. However, electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated responses in wild-type as well as trp and trpl mutant flies were all inhibited by 2-APB. This action of 2-APB is likely InsP(3)R-independent since InsP(3)Rs are dispensable for the light response. We used triple InsP(3)R knockout DT40 chicken B-cells to further assess the role of InsP(3)Rs in SOC activation. (45)Ca(2+) flux analysis revealed that although DT40 wild-type cells retained normal InsP(3)Rs mediating 2-APB-sensitive Ca(2+) release, the DT40InsP(3)R-k/o cells were devoid of functional InsP(3)Rs. Using intact cells, all parameters of Ca(2+) store function and SOC activation were identical in DT40wt and DT40InsP(3)R-k/o cells. Moreover, in both cell lines SOC activation was completely blocked by 2-APB, and the kinetics of action of 2-APB on SOCs (time dependence and IC(50)) were identical. The results indicate that (a) the action of 2-APB on Ca(2+) entry is not mediated by the InsP(3)R and (b) the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.
Highlights
The mechanism for coupling between Ca2؉ stores and store-operated channels (SOCs) is an important but unresolved question
The resulting depletion of Ca2ϩ stored within the endoplasmic reticulum (ER) lumen serves as the primary trigger for a message that is returned to the plasma membrane, resulting in the slow activation of store-operated channels (SOCs), which mediate the process known as capacitative Ca2ϩ entry [3, 5,6,7,8]
Experiments reveal that the activation of both transient receptor potential (TRP) channels and SOCs is prevented by the action of two different membrane-permeant InsP3 receptor antagonists, xestospongin C and 2-aminoethoxydiphenyl borate (2-APB) [17, 21], providing further evidence for the involvement of the InsP3 receptor in coupling to activate both entry channel types
Summary
The mechanism for coupling between Ca2؉ stores and store-operated channels (SOCs) is an important but unresolved question. The results indicate that InsP3Rs do not mediate the action of 2-APB on Ca2ϩ entry, the effects of 2-APB provide evidence for an important similarity in the function of invertebrate TRP channels, mammalian TRP channels, and mammalian store-operated channels.
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