Electrophysiological characterization of store-operated and agonist-induced Ca2+ entry pathways in endothelial cells

In endothelial cells, agonist-induced Ca2+ entry takes place via the store-operated Ca2+ entry pathway and/or via channel(s) gated by second messengers. As cell stimulation leads to both a partial Ca2+ store depletion as well as the production of second messengers, these two pathways are problematic to distinguish. We showed that passive endoplasmic reticulum (ER) depletion by thapsigargin or cell stimulation by histamine activated a similar Ca2+-release-activated Ca2+ current (CRAC)-like current when 10mM Ba2+/2mM Ca2+ was present in the extracellular solution. Importantly, during voltage clamp recordings, histamine stimulation largely depleted the ER Ca2+ store, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba2+ medium. On the contrary, in the presence of 10mM Ca2+, the ER Ca2+ content remained elevated, and histamine induced an outward rectifying current that was inhibited by Ni2+ and KB-R7943, two blockers of the Na+/Ca2+ exchanger (NCX). Both blockers also reduced histamine-induced cytosolic Ca2+ elevation. In addition, removing extracellular Na+ increased the current amplitude which is in line with a current supported by the NCX. These data are consistent with the involvement of the NCX working in reverse mode (Na+ out/Ca2+ in) during agonist-induced Ca2+ entry in endothelial cell

Quantitative proteomic analysis of skeletal muscles from wild type and transgenic mice carrying recessive Ryr1 mutations linked to congenital myopathies

Skeletal muscle is a highly structured and differentiated tissue responsible for voluntary movement and metabolic regulation. Muscles however, are heterogeneous and depending on their location, speed of contraction, fatiguability and function, can be broadly subdivided into fast and slow twitch as well as subspecialized muscles, with each group expressing common as well as specific proteins. Congenital myopathies are a group of non-inflammatory non-dystrophic muscle diseases caused by mutations in a number of genes, leading to a weak muscle phenotype. In most cases specific muscles types are affected, with preferential involvement of fast twitch muscles as well as extraocular and facial muscles. Here we performed relative and absolute quantitative proteomic analysis of EDL, soleus and extraocular muscles from wild type and transgenic mice carrying compound heterozygous mutations in Ryr1 identified in a patient with a severe congenital myopathy. Our quantitative proteomic study shows that recessive Ryr1 mutations not only decrease the content of RyR1 protein in muscle, but also impact the content of many other proteins; in addition, we provide important insight into the pathological mechanism of congenital myopathies linked to mutations in other genes encoding components of the excitation contraction coupling molecular complex

Measurements of the free luminal ER Ca(2+) concentration with targeted "cameleon" fluorescent proteins

The free ER Ca(2+) concentration, [Ca(2+)](ER), is a key parameter that determines both the spatio-temporal pattern of Ca(2+) signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca(2+) activity within the ER is thus critical for our understanding of cell signaling. Such measurements, however, are particularly challenging given the highly dynamic nature of Ca(2+) signals, the complex architecture of the ER, and the difficulty of addressing probes specifically into the ER lumen. Prompted by these challenges, a number of ingenious approaches have been developed over the last years to measure ER Ca(2+) by optical means. The two main strategies used to date are Ca(2+)-sensitive synthetic dyes trapped into organelles and genetically encoded probes, based either on the photoprotein aequorin or on the green fluorescent protein (GFP). The GFP-based Ca(2+) indicators comprise the camgaroo and pericam probes based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. Each approach offers unique advantages and suffers from specific drawbacks. In this review, we will discuss the advantages and pitfalls of using the genetically encoded "cameleon" Ca(2+) indicators for ER Ca(2+) measurements

reserve cell activation

Abstractdata associated with paper published in Cells: activation and migration of human skeletal muscle stem cells in vitro differently rely on calcium signal

STIM1 knockdown reveals that store-operated Ca2+ channels located close to sarco/endoplasmic Ca2+ ATPases (SERCA) pumps silently refill the endoplasmic reticulum

Stromal interaction molecule (STIM) proteins are putative ER Ca2+ sensors that recruit and activate store-operated Ca2+ (SOC) channels at the plasma membrane, a process triggered by the Ca2+ depletion of the endoplasmic reticulum (ER). To test whether STIM1 is required for ER refilling, we used RNA interference and measured Ca2+ signals in the cytosol, the ER, and the mitochondria of HeLa cells. Knockdown of STIM1 (mRNA levels, 73%) reduced SOC entry by 73% when sarco/endoplasmic Ca2+ ATPases (SERCA) were inhibited by thapsigargin but did not prevent Ca2+ stores refilling when cells were stimulated by physiological agonists. Stores could be fully refilled by increasing the external Ca2+ concentration above physiological values, but no cytosolic Ca2+ signals were detected during store refilling even at very high Ca2+ concentrations. [Ca2+](ER) measurements revealed that the basal activity of SERCA was not affected in STIM1 knockdown cells and that [Ca2+](ER) levels were restored within 2 min in physiological saline following store depletion. Mitochondrial inhibitors reduced ER refilling in wild-type but not in STIM1 knockdown cells, indicating that ER refilling does not require functional mitochondria at low STIM1 levels. Our data show that ER refilling is largely preserved at reduced STIM1 levels, despite a drastic reduction of store-operated Ca2+ entry, because Ca2+ ions are directly transferred from SOC channels to SERCA. These findings are consistent with the formation of microdomains containing not only SOC channels on the plasma membrane and STIM proteins on the ER but also SERCA pumps and mitochondria to refill the ER without perturbing the cytosol

Regulation of plasma membrane calcium fluxes by mitochondria

The role of mitochondria in cell signaling is becoming increasingly apparent, to an extent that the signaling role of mitochondria appears to have stolen the spotlight from their primary function as energy producers. In this chapter, we will review the ionic basis of calcium handling by mitochondria and discuss the mechanisms that these organelles use to regulate the activity of plasma membrane calcium channels and transporters

Transient receptor potential canonical channels are required for in vitro endothelial tube formation

In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca(2+) oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells