The Role of the Pore-forming Region in the Regulation of IP3 Receptor by Luminal Ca2+

Abstract

It is well known that submaximal concentrations of IP3 release only a portion of the intracellular Ca2+ store via the IP3 receptor (IP3R), a phenomenon known as “quantal” Ca2+ release. Such quantal behavior of IP3R is thought to be due to the feedback regulation of the channel by luminal Ca2+. A high level of luminal Ca2+ enhances the sensitivity of IP3R to IP3, while a reduced luminal Ca2+ level desensitizes IP3R. Despite its importance, the molecular basis underling the regulation of IP3R by luminal Ca2+ is unknown. Ryanodine receptors (RyRs), another family of intracellular Ca2+ release channels, also exhibit quantal Ca2+ release in response to agonists, and are regulated by luminal Ca2+. We have recently demonstrated that mutations in the TM10 helix (the pore inner helix) of the RyR2 channel markedly alter the sensitivity of the channel to activation by luminal Ca2+. Given the high degree of sequence homology in the channel pore-forming region between RyR and IP3R, we hypothesize that the TM6 helix in IP3R, corresponding to TM10 in RyR, is also important for luminal Ca2+ regulation of IP3R. To test this hypothesis, we have generated a number of mutations in the TM6 of IP3R and established stable, inducible HEK293 cell lines expressing these mutants. By monitoring the ER luminal Ca2+ level using a fluorescent ER Ca2+ sensor protein, D1ER, we found that mutations in TM6 either increase or decrease the rate of IP3-induced Ca2+ release in permeablized mutant cells. These mutations also affect the sensitivity of ATP-triggered Ca2+ release in intact cells. Further studies at the single channel level should provide new insights into the role of the pore-forming region in the luminal Ca2+ regulation of IP3R.