Disrupted Ca2+ homeostasis and immunodeficiency in patients with functional IP3 receptor subtype 3 defects.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

Missense mutations in inositol 1,4,5-trisphosphate receptor type 3 result in leaky Ca2+ channels and activation of store-operated Ca2+ entry.

Mutations in all subtypes of the inositol 1,4,5-trisphosphate receptor Ca2+ release channel are associated with human diseases. In this report, we investigated the functionality of three neuropathy-associated missense mutations in IP3R3 (V615M, T1424M, and R2524C). The mutants only exhibited function when highly over-expressed compared to endogenous hIP3R3. All variants resulted in elevated basal cytosolic Ca2+ levels, decreased endoplasmic reticulum Ca2+ store content, and constitutive store-operated Ca2+ entry in the absence of any stimuli, consistent with a leaky IP3R channel pore. These variants differed in channel function; when stably over-expressed the R2524C mutant was essentially dead, V615M was poorly functional, and T1424M exhibited activity greater than that of the corresponding wild-type following threshold stimulation. These results demonstrate that a common feature of these mutations is decreased IP3R3 function. In addition, these mutations exhibit a novel phenotype manifested as a constitutively open channel, which inappropriately gates SOCE in the absence of stimulation.

CREB regulates the expression of type 1 inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a central role in regulating intracellular Ca2+ signals in response to a variety of internal and external cues. Dysregulation of IP3R signaling is the underlying cause for numerous pathological conditions. It is well established that the activities of IP3Rs are governed by several post-translational modifications, including phosphorylation by protein kinase A (PKA). However, the long-term effects of PKA activation on expression of IP3R subtypes remains largely unexplored. In this report, we investigate the effects of chronic stimulation and tonic activity of PKA on the expression of IP3R subtypes. We demonstrate that expression of the type 1 IP3R (IP3R1) is augmented upon prolonged activation of PKA or upon ectopic overexpression of cyclic AMP-response element-binding protein (CREB) without altering IP3R2 and IP3R3 abundance. By contrast, inhibition of PKA or blocking CREB diminished IP3R1 expression. We also demonstrate that agonist-induced Ca2+-release mediated by IP3R1 is significantly attenuated upon blocking of CREB. Moreover, CREB - by regulating the expression of KRAS-induced actin-interacting protein (KRAP) - ensures correct localization and licensing of IP3R1. Overall, we report a crucial role for CREB in governing both the expression and correct localization of IP3R1. This article has an associated First Person interview with the first author of the paper.