Biology subjects

Terry, L. E.

Publications and source records attributed to Terry, L. E..

5 recordsLinked to original sources

bioRxiv2022

Structural dynamics underlying gating and regulation in IP3R channel

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP in IP3R1, revealing conformational changes that couple ligand-binding to channel opening. Using a GMM based deep learning approach and 3D variability analysis, we extracted dynamic properties of the key protein domains. From this, we find that IP3 binding relies upon intrinsic flexibility of the cytoplasmic ARM2 domain. Our results highlight a key role of dynamic side chains surrounding the ion conduction path in regulating gating behavior of IP3R channels. Altogether, this work defines a structural platform for mechanistic understanding of the molecular dynamics underlying ligand-binding, activation and regulation of the IP3R activity.

biophysics↗

bioRxiv2022

Functional Determination of Calcium Binding Sites Required for the Activation of Inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) initiate a diverse array of physiological responses by carefully orchestrating intracellular calcium (Ca2+) signals in response to various external cues. Notably, IP3R channel activity is determined by several obligatory factors including IP3, Ca2+ and ATP. The critical basic amino acid residues in the N-terminal IP3-binding core (IBC) region that facilitate IP3 binding are well characterized. In contrast, the residues conferring the biphasic regulation by Ca2+ are yet to be ascertained. Using comparative structural analysis of Ca2+ binding sites identified in two main families of intracellular Ca2+-release channels, ryanodine receptors (RyRs) and IP3Rs, we identified putative acidic residues coordinating Ca2+ in the cytosolic calcium sensor region in IP3Rs. We determined the consequences of substituting putative Ca2+ binding, acidic residues in IP3R family members. We show that the agonist-induced Ca2+ release, single channel open probability (P0) and Ca2+ sensitivities are markedly altered when the negative charge on the conserved acidic side chain residues are neutralized. Remarkably, neutralizing the negatively charged side chain on two of the residues individually in the putative Ca2+ binding pocket shifted the Ca2+ required to activate IP3R to higher concentrations, indicating that these residues likely are a component of the Ca2+ activation site in IP3R. Taken together, our findings indicate that Ca2+ binding to a well conserved activation site is a common underlying mechanism resulted in increased channel activity shared by IP3Rs and RyRs.

physiology↗

bioRxiv2021

Functional Communication between IP3R and STIM2 at sub-threshold stimuli is a critical checkpoint for initiation of SOCE

Stromal interaction molecules, STIM1 and STIM2, sense decreases in the endoplasmic reticulum (ER) [Ca2+] ([Ca2+]ER) and cluster in ER-plasma membrane (ER-PM) junctions where they recruit and activate Orai1. While STIM1 responds when [Ca2+]ER is relatively low, STIM2 displays constitutive clustering in the junctions and is suggested to regulate basal Ca2+ entry. The cellular cues that determine STIM2 clustering under basal conditions is not known. By using gene editing to fluorescently tag endogenous STIM2, we report that endogenous STIM2 is constitutively localized in mobile and immobile clusters. The latter associate with ER-PM junctions and recruit Orai1 under basal conditions. Agonist stimulation increases immobile STIM2 clusters which co- ordinate recruitment of Orai1 and STIM1 to the junctions. Extended synaptotagmin (E-Syt)2/3 are required for forming the ER-PM junctions, but are not sufficient for STIM2 clustering. Importantly, inositol 1,4,5-triphosphate receptor (IP3R) function and local [Ca2+]ER are the main drivers of immobile STIM2 clusters. Enhancing, or decreasing, IP3R function at ambient [IP3] causes corresponding increase, or attenuation, of immobile STIM2 clusters. We show that immobile STIM2 clusters denote decreases in local [Ca2+]ER mediated by IP3R that is sensed by the STIM2-N terminus. Finally, under basal conditions, ambient PIP2-PLC activity of the cell determines IP3R function, immobilization of STIM2, and basal Ca2+ entry while agonist stimulation augments these processes. Together, our findings reveal that immobilization of STIM2 clusters within ER-PM junctions, a first response to ER-Ca2+ store depletion, is facilitated by the juxtaposition of IP3R and marks a checkpoint for initiation of Ca2+ entry. SignificanceSTIM proteins sense decreases in [Ca2+]ER and cluster in endoplasmic reticulum (ER)-plasma membrane (PM) junctions where they recruit and activate Orai1. While STIM1 clustering requires substantial [Ca2+]ER decrease, STIM2 displays pre-clustering under resting conditions and regulates basal Ca2+ entry. The mechanism(s) underlying constitutive clustering of STIM2 is not known. We show herein that endogenous STIM2 assembles as mobile and immobile clusters and that Orai1 is recruited to the latter. Anchoring of STIM2 clusters is triggered by decreases in local [Ca2+]ER that is mediated by ambient activity of IP3R and sensed by the STIM2 N-terminus. This functional link between IP3R and STIM2 governs constitutive STIM2 clustering and ensures coupling of [Ca2+]ER decrease at sub-threshold stimuli with activation of Ca2+ entry.

physiology↗

bioRxiv2021

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 calcium signals in response to a variety of internal/external cues. Dysregulation of IP3R signaling is the underlying cause for numerous pathological conditions. It is also well established that the activity of IP3Rs is 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 sub-types, remains largely unexplored. In this report, we investigate the effect of chronic activation of PKA on expression of IP3R sub-types. We demonstrate that expression of IP3R1 is augmented upon prolonged activation of PKA or upon ectopic over-expression of CREB but does not alter IP3R2 and IP3R3 sub-type abundance. Conversely, inhibition of PKA or blocking endogenous CREB diminished IP3R1 expression. We also demonstrate that agonist-induced Ca2+-release mediated by IP3R1 is significantly attenuated upon blocking endogenous CREB. Moreover, CREB by regulating the expression of KRAS-induced actin-interacting protein (KRAP) ensures proper localization and licensing of IP3R1. Overall, we report a crucial role for CREB in governing both the expression and proper localization of IP3R1. Summary statementWe report a critical role of CREB in regulating the expression and proper localization of IP3R1. Agonist-induced Ca2+ release and Ca2+ puffs generated by IP3R1 are diminished upon blocking the function of endogenous CREB.

physiology↗

bioRxiv2018

Acetylcholine inhibits platelet activation and regulates hemostasis

Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but there are fewer identified endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO). Acetylcholinesterase inhibitors such as donepezil can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets.\n\nWe measured the effect of acetylcholine or analogues of acetylcholine upon human platelet activation ex vivo. We characterized expression of components of the acetylcholine signaling pathway in human platelets. We tested the effect of a subunit of the acetylcholine receptor, CHRNA7, on acetylcholine signaling in platelets. Acetylcholine and analogues of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and GPIIbIIIA conformational changes. Conversely, we found that antagonists of the acetylcholine receptor such as pancuronium enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase such as donepezil also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Human platelets express members of the acetylcholine signaling pathway including CHRNA2, CHRNA7, CHRNB1, and ACHE. Platelets from mice lacking Chrna7 are hyperactive when stimulated by thrombin and resistant to inhibition by acetylcholine. Furthermore, acetylcholinesterase inhibitors prolonged bleeding in wild-type mice. Knockout mice lacking Chrna7 subunits of the acetylcholine receptor display prolonged bleeding as well.\n\nOur data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for anti-thrombotic therapies.

pharmacology and toxicology↗