Regulation of store-operated Ca2+ entry by IP3 receptors independent of their ability to release Ca2+
Curation statements for this article:-
Curated by eLife
eLife assessment
In this manuscript, Chakraborty et al address the role of IP3R1 in regulating store-operated calcium entry in neurons and neural progenitors. Long-standing observations in non-neuronal cells have shown that IP3Rs are not required for SOCE. In contrast to those findings, this manuscript determines that in neuronal cells, knockdown of IP3R1 suppresses SOCE by disrupting ER-plasma membrane contact sites. The paper supports a novel role for IP3R1 as a tether in promoting membrane contact sites which would have broad implications for a range of physiological processes including SOCE and lipid metabolism.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Loss of endoplasmic reticular (ER) Ca 2+ activates store-operated Ca 2+ entry (SOCE) by causing the ER localized Ca 2+ sensor STIM to unfurl domains that activate Orai channels in the plasma membrane at membrane contact sites (MCS). Here, we demonstrate a novel mechanism by which the inositol 1,4,5 trisphosphate receptor (IP 3 R), an ER-localized IP 3 -gated Ca 2+ channel, regulates neuronal SOCE. In human neurons, SOCE evoked by pharmacological depletion of ER-Ca 2+ is attenuated by loss of IP 3 Rs, and restored by expression of IP 3 Rs even when they cannot release Ca 2+ , but only if the IP 3 Rs can bind IP 3 . Imaging studies demonstrate that IP 3 Rs enhance association of STIM1 with Orai1 in neuronal cells with empty stores; this requires an IP 3 -binding site, but not a pore. Convergent regulation by IP 3 Rs, may tune neuronal SOCE to respond selectively to receptors that generate IP 3 .
Article activity feed
-
eLife assessment
In this manuscript, Chakraborty et al address the role of IP3R1 in regulating store-operated calcium entry in neurons and neural progenitors. Long-standing observations in non-neuronal cells have shown that IP3Rs are not required for SOCE. In contrast to those findings, this manuscript determines that in neuronal cells, knockdown of IP3R1 suppresses SOCE by disrupting ER-plasma membrane contact sites. The paper supports a novel role for IP3R1 as a tether in promoting membrane contact sites which would have broad implications for a range of physiological processes including SOCE and lipid metabolism.
-
Reviewer #1 (Public Review):
This paper describes a novel and important role for IP3 receptors (IP3R) in the control of store-operated calcium entry (SOCE) in neurons. The authors provide strong evidence that in human neural progenitor cells before and after differentiation in vitro, as well as a neuroblastoma cell line (SH-SY5Y), knockdown of the IP3R1 isoform significantly diminishes SOCE triggered by ER calcium store depletion. Interestingly, SOCE is fully restored in these cells by overexpressing WT IP3R1 or a mutant that cannot conduct Ca2+ but is not restored by an IP3R1 mutant that cannot bind IP3. Based on these results the authors conclude that IP3-bound IP3R1 enhances SOCE not by depleting ER Ca2+ but through an as yet uncharacterized physical interaction.
The authors propose that resting levels of IP3 are sufficient for this …
Reviewer #1 (Public Review):
This paper describes a novel and important role for IP3 receptors (IP3R) in the control of store-operated calcium entry (SOCE) in neurons. The authors provide strong evidence that in human neural progenitor cells before and after differentiation in vitro, as well as a neuroblastoma cell line (SH-SY5Y), knockdown of the IP3R1 isoform significantly diminishes SOCE triggered by ER calcium store depletion. Interestingly, SOCE is fully restored in these cells by overexpressing WT IP3R1 or a mutant that cannot conduct Ca2+ but is not restored by an IP3R1 mutant that cannot bind IP3. Based on these results the authors conclude that IP3-bound IP3R1 enhances SOCE not by depleting ER Ca2+ but through an as yet uncharacterized physical interaction.
The authors propose that resting levels of IP3 are sufficient for this activity, based on the ability of a Gq inhibitor to mimic the effect of IP3R1 knockdown on SOCE. Importantly, the inhibitor does not affect SOCE in cells lacking IP3R1, arguing against a nonspecific effect of the drug. The ability of partial binding of low levels of IP3 to support this activity is somewhat surprising, and further studies will be needed to test whether the enhancing effect is amplified by receptor-driven elevation of IP3.
An important question is how the IP3R1 acts to enhance SOCE. A proximity ligation assay clearly showed that IP3R1 knockdown disrupted STIM1 and Orai1 colocalization after store depletion, supporting the notion that IP3R1 acts to enhance STIM1-Orai1 interactions. How might this occur? The authors suggest that IP3R1 enhances the formation or stability of ER-plasma membrane (ER-PM) junctions where STIM1 and Orai1 combine to trigger SOCE, based on the rescue of SOCE by overexpression of STIM1 or E-syt1, both of which promote ER-PM junction formation or stability. However, this is indirect evidence, and a more direct demonstration of how IP3R1 affects ER-PM junction abundance and size would add stronger support for this hypothesis.
The authors suggest that the effects of IP3R1 described here may serve to selectively promote SOCE in response to stimuli that generate IP3 as opposed to other signals that release ER Ca2+. This proposal and its functional impact need further study, including why it appears to be cell-specific, occurring in neurons but not HEK 293 cells and other cell types.
-
Reviewer #2 (Public Review):
Chakraborty et al. present a comprehensive analysis of the role of the IP3R in regulating SOCE in neuronal cells starting with human neurons derived from stem cells and continuing with SH-SY5Y cells after careful characterization of the maintenance of the inhibitory role of IP3R. They also show differential effects in non-neuronal cell lines. The work is careful and the data convincing. The conclusion that IP3Rs somehow stabilize ER-PM MCS to enhance SOCE is supported by the findings especially the surprising finding that the IP3R effect does not require a functional pore but does require IP3 binding to IP3R. Overall this is a careful, well-done analysis. However, the conclusion that IP3R stabilizes ER-PM MCS is mostly inferred from the current data. The authors need to extend the finding by directly …
Reviewer #2 (Public Review):
Chakraborty et al. present a comprehensive analysis of the role of the IP3R in regulating SOCE in neuronal cells starting with human neurons derived from stem cells and continuing with SH-SY5Y cells after careful characterization of the maintenance of the inhibitory role of IP3R. They also show differential effects in non-neuronal cell lines. The work is careful and the data convincing. The conclusion that IP3Rs somehow stabilize ER-PM MCS to enhance SOCE is supported by the findings especially the surprising finding that the IP3R effect does not require a functional pore but does require IP3 binding to IP3R. Overall this is a careful, well-done analysis. However, the conclusion that IP3R stabilizes ER-PM MCS is mostly inferred from the current data. The authors need to extend the finding by directly assessing the size, density, and the number of ER-PM MCS using endogenous STIM1 (there are reliable antibodies for STIM1) to confirm their conclusion that when IP3R is knocked down ER-PM MCS are smaller/less dense. Another interesting experiment that would support their conclusion is expressing tagged STIM1 and Orai1 and observing their interaction in real time after store depletion. These experiments would need to be carefully controlled to select cells with low levels of expression of STIM1-Orai1 as there are hints from their current data that high expressors would not exhibit the IP3R dependence on SOCE. So, some independent experimental evidence that IP3R knockdown is affecting ER-PM MCS and not STIM1-Orai1 interaction directly to support the presented PLA data would greatly support the final conclusion of the paper. From the PLA assay alone it is difficult to differentiate between poor direct STIM1-Orai1 interaction versus stability of ER-PM MCS.
-
Reviewer #3 (Public Review):
SOCE is a ubiquitous cell signalling pathway that sustains long-lasting Ca2+ elevations required for the proliferation of T cells and the differentiation and contractility of skeletal muscle. Patients with loss of function mutations in either STIM1 or ORAI1 suffer from severe combined immunodeficiency while patients with gain-of-function mutations suffer from muscle weakness. The report that an intracellular calcium channel acts as a tether at membrane contact sites to regulate the activity of STIM/ORAI channels is thus relevant for health and disease, given the essential role of the SOCE pathway for immune and muscle cell function.
The IP3R is the major Ca2+ release pathway that initiates the STIM/ORAI activation cascade and the group of Colin Taylor (coauthor of the present study) showed that a pool of …
Reviewer #3 (Public Review):
SOCE is a ubiquitous cell signalling pathway that sustains long-lasting Ca2+ elevations required for the proliferation of T cells and the differentiation and contractility of skeletal muscle. Patients with loss of function mutations in either STIM1 or ORAI1 suffer from severe combined immunodeficiency while patients with gain-of-function mutations suffer from muscle weakness. The report that an intracellular calcium channel acts as a tether at membrane contact sites to regulate the activity of STIM/ORAI channels is thus relevant for health and disease, given the essential role of the SOCE pathway for immune and muscle cell function.
The IP3R is the major Ca2+ release pathway that initiates the STIM/ORAI activation cascade and the group of Colin Taylor (coauthor of the present study) showed that a pool of immobile receptors licensed to respond to physiological stimuli localizes near STIM-ORAI interaction sites at ER-PM junctions DOI: 10.1016/j.ceb.2018.10.001. This group further showed that IP3Rs are tethered to PM-bound actin by the KRas-induced actin-interacting protein (KRAP) DOI: 10.1038/s41467-021-24739-9 while the group of Indu Ambudkar showed that IP3R is juxtaposed to immobile STIM2 clusters within ER-PM junctions DOI: https://doi.org/10.1073/pnas.2114928118 The mechanism by which IP3R impinges on SOCE at ER-PM contact sites remains unclear, however.
The present study provides an important clue by showing that IP3Rs themselves can act as tethering proteins independently of their calcium release function. However, several important questions remain unanswered. Are the native and mutated receptors recruited differentially to ER-PM junctions? If so, what interacting partner(s) and mechanisms enable IP3-bound receptors to enhance the interactions between STIM1 and ORAI1? And why is this effect restricted to neuronal cells?
Previous studies indicate that IP3R can interact with actin via KRAP, with STIM proteins, with ORAI channels, and with phosphoinositides. The authors point to phosphoinositides as a potential target that could explain the need for IP3, but this possibility has not been experimentally addressed. They should establish whether phosphoinositides are involved in the recruitment of IP3R receptors and provide additional mechanistic insight by documenting whether IP3R depletion impacts the stability of contact sites or their ability to exchange lipids between membranes. Another unresolved question relates to the observation that the phenotype is restricted to neuronal cell types and absent in HEK-293 cells typically used for electrophysiological recordings of CRAC currents. The authors should attempt to clarify the molecular basis of this difference between cell types.
From a methodological standpoint, one limitation is that the functional assays used are quite indirect. One critical SOCE determinant is the filling state of intracellular calcium stores, which was estimated indirectly by measuring the amplitude of the Ca2+ elevation evoked by the addition of the SERCA inhibitor thapsigargin. Although this method is widely used it does not directly reflect the key parameter driving STIM1 activation which is the free calcium concentration within the ER lumen. Direct ER [Ca2+] recordings are required to clarify this critical point.
-
-
-
-