Regulation Of Inositol 1,4,5-Trisphosphate Receptor Isoforms By O-Linked Glycosylation

Abstract

as ''quantal'' Ca2þ release. Such quantal behavior of IP3R is thought to be due to the feedback regulation of the channel by luminal Ca2þ. A high level of luminal Ca2þ enhances the sensitivity of IP3R to IP3, while a reduced luminal Ca2þ level desensitizes IP3R. Despite its importance, the molecular basis underling the regulation of IP3R by luminal Ca2þ is unknown. Ryanodine receptors (RyRs), another family of intracellular Ca2þ release channels, also exhibit quantal Ca2þ release in response to agonists, and are regulated by luminal Ca2þ. 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 Ca2þ. 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 Ca2þ 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 Ca2þ level using a fluorescent ER Ca2þ sensor protein, D1ER, we found that mutations in TM6 either increase or decrease the rate of IP3-induced Ca2þ release in permeablized mutant cells. These mutations also affect the sensitivity of ATP-triggered Ca2þ 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 Ca2þ regulation of IP3R.

Type 3 Inositol 1,4,5‐Trisphosphate Receptor Is Increased and Enhances Malignant Properties in Cholangiocarcinoma

“…Calcium signals may be increased not only as a result of increased ITPR expression but also because of increased channel activity, and O-linked glycosylation enhances calcium release from ITPR3. (25) Therefore, ITPR3 was immunoprecipitated from MzCha1, HuCCT1, and NHC cells, and the immunoprecipitated ITPR3 was probed for O-GlcNAc modification. There was no significant difference in the fraction of ITPR3 that was O-glycosylated between NHC cells and either of the two CCA cell lines, suggesting that modulation of this modification does not contribute to enhanced calcium signaling in CCA (Supporting Fig.…”