Jonathan Pacheco

Direct Binding Between Orai1 and AC8 Mediates Dynamic Interplay Between Ca2+ and cAMP Signaling

A signaling complex enables the compartmentalized regulation of cyclic AMP signaling by calcium entering through a specific channel. Bound to Signal in Close Quarters Interplay between the calcium and the cyclic adenosine monophosphate (cAMP) signaling pathways is crucial to numerous physiological events. Although membrane-bound calcium-sensitive adenylyl cyclases (ACs) are sensitive to submicromolar concentrations of calcium in vitro, in cells they are highly selective in responding to store-operated calcium (SOC) entry rather than to calcium released from intracellular stores or entering the cell through ionophores. Here, Willoughby et al. used a combination of live-cell imaging techniques and biochemical approaches to resolve this conundrum and showed that AC8, which is stimulated by calcium-bound calmodulin, forms a direct protein-protein interaction with Orai1, the pore-forming component of the channel that mediates SOC entry. The existence of AC8 in a complex with SOC channels provides a mechanism for the compartmentalized regulation of cAMP signaling by specific subcellular calcium signals. The interplay between calcium ion (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling underlies crucial aspects of cell homeostasis. The membrane-bound Ca2+-regulated adenylyl cyclases (ACs) are pivotal points of this integration. These enzymes display high selectivity for Ca2+ entry arising from the activation of store-operated Ca2+ (SOC) channels, and they have been proposed to functionally colocalize with SOC channels to reinforce crosstalk between the two signaling pathways. Using a multidisciplinary approach, we have identified a direct interaction between the amino termini of Ca2+-stimulated AC8 and Orai1, the pore component of SOC channels. High-resolution biosensors targeted to the AC8 and Orai1 microdomains revealed that this protein-protein interaction is responsible for coordinating subcellular changes in both Ca2+ and cAMP. The demonstration that Orai1 functions as an integral component of a highly organized signaling complex to coordinate Ca2+ and cAMP signals underscores how SOC channels can be recruited to maximize the efficiency of the interplay between these two ubiquitous signaling pathways.

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.

The P2X7/P2X4 interaction shapes the purinergic response in murine macrophages.

The ATP-gated P2X4 and P2X7 receptors are cation channels, co-expressed in excitable and non-excitable cells and play important roles in pain, bone development, cytokine release and cell death. Although these receptors interact the interacting domains are unknown and the functional consequences of this interaction remain unclear. Here we show by co-immunoprecipitation that P2X4 interacts with the C-terminus of P2X7 and by fluorescence resonance energy transfer experiments that this receptor-receptor interaction is driven by ATP. Furthermore, disrupting the ATP-driven interaction by knocking-out P2X4R provoked an attenuation of P2X7-induced cell death, dye uptake and IL-1β release in macrophages. Thus, P2X7 interacts with P2X4 via its C-terminus and disrupting the P2X7/P2X4 interaction hinders physiological responses in immune cells.

Combined single channel and single molecule detection identifies subunit composition of STIM1-activated transient receptor potential canonical (TRPC) channels.

Depletion of intracellular calcium ion stores initiates a rapid cascade of events culminating with the activation of the so-called Store-Operated Channels (SOC) at the plasma membrane. Calcium influx via SOC is essential in the initiation of calcium-dependent intracellular signaling and for the refilling of internal calcium stores, ensuring the regeneration of the signaling cascade. In spite of the significance of this evolutionary conserved mechanism, the molecular identity of SOC has been the center of a heated controversy spanning over the last 20 years. Initial studies positioned some members of the transient receptor potential canonical (TRPC) channel superfamily of channels (with the more robust evidence pointing to TRPC1) as a putative SOC. Recent evidence indicates that Stromal Interacting Molecule 1 (STIM1) activates some members from the TRPC family of channels. However, the exact subunit composition of TRPC channels remains undetermined to this date. To identify the subunit composition of STIM1-activated TRPC channels, we developed novel method, which combines single channel electrophysiological measurements based on the patch clamp technique with single molecule fluorescence imaging. We termed this method Single ion Channel Single Molecule Detection technique (SC-SMD). Using SC-SMD method, we have obtained direct evidence of the subunit composition of TRPC channels activated by STIM1. Furthermore, our electrophysiological-imaging SC-SMD method provides evidence at the molecular level of the mechanism by which STIM1 and calmodulin antagonize to modulate TRPC channel activity.

A cholesterol-binding domain in STIM1 modulates STIM1-Orai1 physical and functional interactions.

STIM1 and Orai1 are the main components of a widely conserved Calcium influx pathway known as store-operated calcium entry (SOCE). STIM1 is a calcium sensor, which oligomerizes and activates Orai channels when calcium levels drop inside the endoplasmic reticulum (ER). The series of molecular rearrangements that STIM1 undergoes until final activation of Orai1 require the direct exposure of the STIM1 domain known as SOAR (Stim Orai Activating Region). In addition to these complex molecular rearrangements, other constituents like lipids at the plasma membrane, play critical roles orchestrating SOCE. PI(4,5)P2 and enriched cholesterol microdomains have been shown as important signaling platforms that recruit the SOCE machinery in steps previous to Orai1 activation. However, little is known about the molecular role of cholesterol once SOCE is activated. In this study we provide clear evidence that STIM1 has a cholesterol-binding domain located inside the SOAR region and modulates Orai1 channels. We demonstrate a functional association of STIM1 and SOAR to cholesterol, indicating a close proximity of SOAR to the inner layer of the plasma membrane. In contrast, the depletion of cholesterol induces the SOAR detachment from the plasma membrane and enhances its association to Orai1. These results are recapitulated with full length STIM1.

Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca2+ leak using synthetic Ca2+ indicators that report changes in both the cytoplasmic ([Ca2+]i) and the luminal ER ([Ca2+]ER) Ca2+ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca2+ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca2+]ER but did not decrease the ER Ca2+ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP3R and only Orai3 channel supported the 2-APB-induced ER Ca2+ leak, while Orai1 and Orai2 inhibited this type of ER Ca2+ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca2+ leak but did not modify the ER Ca2+ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca2+]i response after TG but only when the ER store had been overloaded with Ca2+ by eliminating the acidic internal Ca2+ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca2+ leak but forms an ER Ca2+ leak channel that is limiting the overloading with Ca2+ of the ER store.

Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains

Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE. In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern (unobstructed diffusion) opposite to basal cholesterol conditions were most of Orai1 channels moves in a confined space, as assessed by Fluorescence Correlation Spectroscopy, Cav1 overexpression inhibited these alterations maintaining Orai1 into a confined and partially confined movement. These results not only highlight the complex effect of cholesterol regulation on SOCE, but also indicate a direct regulatory effect on Orai1 localization and compartmentalization by this lipid.

Microdomains Associated to Lipid Rafts

Store Operated Ca(2+) Entry (SOCE), the main Ca(2+) influx mechanism in non-excitable cells, is implicated in the immune response and has been reported to be affected in several pathologies including cancer. The basic molecular constituents of SOCE are Orai, the pore forming unit, and STIM, a multidomain protein with at least two principal functions: one is to sense the Ca(2+) content inside the lumen of the endoplasmic reticulum(ER) and the second is to activate Orai channels upon depletion of the ER. The link between Ca(2+) depletion inside the ER and Ca(2+) influx from extracellular media is through a direct association of STIM and Orai, but for this to occur, both molecules have to interact and form clusters where ER and plasma membrane (PM) are intimately apposed. In recent years a great number of components have been identified as participants in SOCE regulation, including regions of plasma membrane enriched in cholesterol and sphingolipids, the so called lipid rafts, which recruit a complex platform of specialized microdomains, which cells use to regulate spatiotemporal Ca(2+) signals.

A Microscopic View of the Store-Operated Calcium Entry-Pathway

Orai and STIM are the basic components of a highly complex and regulated mechanism for Ca2+ entry into the cell, known as store-operated calcium entry (SOCE). The activation of plasma membrane G-protein-coupled receptors associated with the phospholipase C cascade results in the rapid and massive production of inositol 1,4,5-triphosphate (IP3). This second messenger triggers the massive efflux of Ca2+ from the endoplasmic reticulum and into the cytosol, resulting in the oligomerization of the stromal interacting molecule (STIM1), a sensor of ER Ca2+. STIM1 oligomers (the so-called puncta) activate Orai channels at the plasma membrane, triggering the influx of Ca2+ into the cytosol. Several microscopy techniques have been implemented to study SOCE, resulting in stunning images of protein complexes assembling in real time. However, little attention has been paid to the findings about this complex mechanism from the imaging point of view, some of which appear to produce contradictory results. In the present review we gathered all the information about SOCE obtained with imaging techniques and contrast these findings with those obtained with alternative methods.

STIM-TRP Pathways and Microdomain Organization: Auxiliary Proteins of the STIM/Orai Complex

The basic paradigm of a mechanism for calcium influx triggered after a reduction on calcium store content implies a sensor of calcium concentration on the endoplasmic reticulum (the stores) and a calcium channel immersed on the plasma membrane. These two basic components are STIM and Orai, the most fundamental and minimal molecular constituents of the store-operated calcium entry mechanism. However, even when minimal components can be reduced to these two proteins, the intricate process involved in approximating two cellular membranes (endoplasmic reticulum, ER and plasma membrane, PM) require the participation of several other components, many of which remain unidentified to this date. Here we review several of the proteins identified as constituents of the so-called store-operated calcium influx complex (SOCIC) and discuss their role in modulating this complex phenomenon.