Abstract
Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.
Highlights
The second messenger inositol [1,4,5]-trisphosphate (Ins[1,4,5]P3)1 binds to Ins[1,4,5]P3 receptors in the endoplasmic reticulum, causing release of stored Ca2ϩ into the cell
We provide evidence that luminal Ca2؉ is exerting its effects via a direct interaction with the luminal face of the receptor
Interpretation of the data is complicated by the possibility of interactions of the divalent cations used as current carriers with potential Ca2ϩ binding sites on the luminal face of the receptor
Summary
The second messenger inositol [1,4,5]-trisphosphate (Ins[1,4,5]P3) binds to Ins[1,4,5]P3 receptors in the endoplasmic reticulum, causing release of stored Ca2ϩ into the cell. Bezprozvanny and Ehrlich [37] investigated the effects of trans (or intraluminal) Ca2ϩ using cerebellar microsomes fused to planar lipid bilayers, with Sr2ϩ, Ba2ϩ, or Mg2ϩ as current carrier (the use of Kϩ was precluded by the presence of Kϩ channels in the microsomal membranes). Interpretation of the data is complicated by the possibility of interactions of the divalent cations used as current carriers with potential Ca2ϩ binding sites on the luminal face of the receptor To resolve this complication, we have used purified cerebellar Type 1 receptor inlaid into bilayers prepared by the method of Schindler [38]. The detailed channel parameters that we describe strongly support the idea that channel behavior is dependent on luminal Ca2ϩ concentrations and that, the receptor is a potential transducer for the filling state of intracellular Ca2ϩ stores
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