Raphael Courjaret

Mid-range Ca2+ signalling mediated by functional coupling between store-operated Ca2+ entry and IP3-dependent Ca2+ release

The versatility and universality of Ca(2+) signals stem from the breadth of their spatial and temporal dynamics. Spatially, Ca(2+) signalling is well studied in the microdomain scale, close to a Ca(2+) channel, and at the whole-cell level. However, little is known about how local Ca(2+) signals are regulated to specifically activate spatially distant effectors without a global Ca(2+) rise. Here we show that an intricate coupling between the inositol 1,4,5 trisphosphate (IP3) receptor, SERCA pump and store-operated Ca(2+) entry (SOCE) allows for efficient mid-range Ca(2+) signalling. Ca(2+) flowing through SOCE is taken up into the ER lumen by the SERCA pump, only to be re-released by IP3Rs to activate distal Ca(2+)-activated Cl(-) channels (CaCCs). This CaCC regulation contributes to setting the membrane potential of the cell. Hence functional coupling between SOCE, SERCA and IP3R limits local Ca(2+) diffusion and funnels Ca(2+) through the ER lumen to activate a spatially separate Ca(2+) effector.

STIM and Orai in cellular proliferation and division.

Cellular proliferation and division are central processes in the development, survival and evolution of living systems. Transitioning into the cell division phase of the cell cycle encompasses dramatic remodeling of cellular organelles and signaling modules including Ca2+ signaling. As well, Ca2+ signals play important roles during progression through various stages of the cell cycle. A ubiquitous Ca2+ influx pathway that is activated based on intracellular Ca2+ store content is store-operated Ca2+ entry (soce). SOCE is activated through a complex interplay between a Ca2+ channel at the cell membrane, Orai1, and a Ca2+ sensor that localizes to the endoplasmic reticulum, STIM1. Herein, we discuss potential roles and regulation of STIM and Orai proteins during cellular proliferation.

Role of the STIM1 C-terminal domain in STIM1 clustering.

Store-operated Ca2+ entry (SOCE) represents a ubiquitous Ca2+ influx pathway activated by the filling state of intracellular Ca2+ stores. SOCE is mediated by coupling of STIM1, the endoplasmic reticulum Ca2+ sensor, to the Orai1 channel. SOCE inactivates during meiosis, partly because of the inability of STIM1 to cluster in response to store depletion. STIM1 has several functional domains, including the Orai1 interaction domain (STIM1 Orai Activating Region (SOAR) or CRAC Activation Domain (CAD)) and STIM1 homomerization domain. When Ca2+ stores are full, these domains are inactive to prevent constitutive Ca2+ entry. Here we show, using the Xenopus oocyte as an expression system, that the C-terminal 200 residues of STIM1 are important to maintain STIM1 in an inactive state when Ca2+ stores are full, through predicted intramolecular shielding of the active STIM1 domains (SOAR/CAD and STIM1 homomerization domain). Interestingly, our data argue that the C-terminal 200 residues accomplish this through a steric hindrance mechanism because they can be substituted by GFP or mCherry while maintaining all aspects of STIM1 function. We further show that STIM1 clustering inhibition during meiosis is independent of the C-terminal 200 residues.

Polymorphism in Endothelial Connexin40 Enhances Sensitivity to Intraluminal Pressure and Increases Arterial Stiffness

Objective—To determine whether impairment of endothelial connexin40 (Cx40), an effect that can occur in hypertension and aging, contributes to the arterial dysfunction and stiffening in these conditions. Approach and Results—A new transgenic mouse strain, expressing a mutant Cx40, (Cx40T202S), specifically in the vascular endothelium, has been developed and characterized. This mutation produces nonfunctional hemichannels, whereas gap junctions containing the mutant are electrically, but not chemically, patent. Mesenteric resistance arteries from Cx40T202S mice showed increased sensitivity of the myogenic response to intraluminal pressure in vitro, compared with wild-type mice, whereas transgenic mice overexpressing native Cx40 (Cx40Tg) showed reduced sensitivity. In control and Cx40Tg mice, the sensitivity to pressure of myogenic constriction was modulated by both NO and endothelium-derived hyperpolarization; however, the endothelium-derived hyperpolarization component was absent in Cx40T202S arteries. Analysis of passive mechanical properties revealed that arterial stiffness was enhanced in vessels from Cx40T202S mice, but not in wild-type or Cx40Tg mice. Conclusions—Introduction of a mutant form of Cx40 in the endogenous endothelial Cx40 population prevents endothelium-derived hyperpolarization activation during myogenic constriction, enhancing sensitivity to intraluminal pressure and increasing arterial stiffness. We conclude that genetic polymorphisms in endothelial Cx40 can contribute to the pathogenesis of arterial disease.

Purinergic Modulation of Granule Cells

Extracellular purines exert their action in the nervous system through purinergic neurotransmission and neuromodulatory processes. Among brain areas, efforts have been made to investigate the purinergic modulation of the cerebellar cortex. In addition, the use of granule cells in culture as a neuronal in vitro model provided important information about the implications of purines in mechanisms such as cell survival and differentiation. This short review is focused on the function of purines in the physiology of granule cells in situ and in vitro. In situ, adenosine has been shown to inhibit some of the glutamatergic and GABAergic synaptic inputs to granule cells. The inhibition of GABA input allows an increase in the excitability of the cell while the output (parallel fibers) of granule cells is also down-regulated by adenosine, suggesting a complex mode of regulation by purines. In vitro, granule cells have been shown to express members of all classes of purinergic receptors, P2X (ionotropic), P2Y (metabotropic) and adenosine receptors. The specific expression of these receptors and the downstream signaling pathways coupling them to cell survival and growth have been extensively studied.

Ca2+ tunnelling through the ER lumen as a mechanism for delivering Ca2+ entering via store‐operated Ca2+ channels to specific target sites

Ca2+ signalling is perhaps the most universal and versatile mechanism regulating a wide range of cellular processes. Because of the many different calcium‐binding proteins distributed throughout cells, signalling precision requires localized rises in the cytosolic Ca2+ concentration. In electrically non‐excitable cells, for example epithelial cells, this is achieved by primary release of Ca2+ from the endoplasmic reticulum via Ca2+ release channels placed close to the physiological target. Because any rise in the cytosolic Ca2+ concentration activates Ca2+ extrusion, and in order for cells not to run out of Ca2+, there is a need for compensatory Ca2+ uptake from the extracellular fluid. This Ca2+ uptake occurs through a process known as store‐operated Ca2+ entry. Ideally Ca2+ entering the cell should not diffuse to the target site through the cytosol, as this would potentially activate undesirable processes. Ca2+ tunnelling through the lumen of the endoplasmic reticulum is a mechanism for delivering Ca2+ entering via store‐operated Ca2+ channels to specific target sites, and this process has been described in considerable detail in pancreatic acinar cells and oocytes. Here we review the most important evidence and present a generalized concept.

The Xenopus TRPV6 homolog encodes a Mg2+-permeant channel that is inhibited by interaction with TRPC1

The TRP gene family encodes primarily cation non‐selective, Ca2+ permeant channels that are involved in a dizzying array of sensory mechanisms. Two channels in this large family TRPV5 and TRPV6 are highly Ca2+ selective and are expressed in epithelia where they are important in Ca2+ uptake. TRPV5/6 are constitutively active, yet the mechanisms regulating their activation in native tissue remains elusive. Here we functionally characterize the Xenopus TRPV6 homolog. xTRPV6 is expressed in the oocyte and encodes a channel that is permeant to divalents including Ca2+, and displays a high permeability to Mg2+. The oocyte does not exhibit functional TRPV6‐like current at rest, showing that the endogenous channel is somehow maintained in an inactive state. We show that endogenous as well as overexpressed xTRPV6 interacts with xTRPC1 and that this interaction inhibits xTRPV6 currents. As such TRPC1 is likely to regulate the activity of TRPV6 under physiological conditions. J. Cell. Physiol. 228: 2386–2398, 2013.

Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity

Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway. Highlighted article: In vivo detection of cAMP and PKA levels in real time reveals that Xenopus oocyte meiotic arrest following progesterone release occurs without any reduction in cAMP levels or PKA activity.

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell–derived cardiomyocytes

BACKGROUND Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. METHODS We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4+ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. RESULTS After 8 days in culture, 94% ± 6% of the NKX2-5GFP+ cells were beating when hESCs embryonic bodies were plated on E4+ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP+ cardiomyocytes were close to the E4+ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. CONCLUSIONS The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony.

Store Operated Ca2+ Entry in Oocytes Modulates the Dynamics of IP3‐Dependent Ca2+ Release from Oscillatory to Tonic

Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store‐operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low‐amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3R) results in a pump‐leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER‐plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3Rs at distant sites, a process we refer to as “Ca2+ teleporting.” Therefore, sustained SOCE activation in the presence of an IP3‐dependent “leak” pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 232: 1095–1103, 2017.