Luis Vaca

STIM1 converts TRPC1 from a receptor-operated to a store-operated channel: Moving TRPC1 in and out of lipid rafts

While the role of members from the TRPC family of channels as receptor-operated channels (ROC) is well established and supported by numerous studies, the role of this family of channels as store-operated channels (SOC) has been the focus of a heated controversy over the last few years. In the present study, we have explored the modulation of STIM1 on human TRPC1 channel. We show that the association of STIM1 to TRPC1 favors the insertion of TRPC1 into lipid rafts, where TRPC1 functions as a SOC. In the absence of STIM1, TRPC1 associates to other members from the TRPC family of channels to form ROCs. A novel TIRFM-FRET method illustrates the relevance of the dynamic association between STIM1 and TRPC1 for the activation of SOC and the lipid raft localization of the STIM1-TRPC1 complex. This study provides new evidence about the dual activity of TRPC1 (forming ROC or SOC) and the partners needed to determine TRPC1 functional fate. It highlights also the role of plasma membrane microdomains and ER-PM junctions in modulating TRPC1 channel function and its association to STIM1.

SOCIC: the store-operated calcium influx complex.

Depletion of intracellular calcium stores via activation of G-protein-coupled receptors associated to the inositol trisphosphate cascade, or by the blockade of the endoplasmic reticulum calcium APTase (SERCA) results in the activation of calcium influx via the so-called store-operated channels (SOCs). The recent identification of STIM1 as the putative sensing molecule responsible for communicating the depleted state of intracellular calcium stores to the plasma membrane channel highlights the relevance of protein complexes in calcium signaling. Further developments in this area identify Orai as part of the store-operated channel complex. Upon depletion of intracellular calcium stores, STIM1 (at the ER) and Orai (at the plasma membrane) aggregate into macromolecular complexes. This molecular aggregation appears to be necessary to induce activation of calcium influx. Several studies have identified novel members from what I would like to define here as the store-operated calcium influx complex (SOCIC), such as the TRPC1 channel, SERCA and the microtubule end tracking protein, EB1. An orchestrated series of events involving the association and dissociation of several protein complexes culminate with the activation of calcium influx upon depletion of the ER. There are other likely players in this sophisticated signaling mechanism, waiting to be uncovered. The SOCIC assembly does not appear to occur in random areas of the plasma membrane, but rather in highly specialized areas known as lipid raft domains. These results strongly suggest that not only proteins but lipids also may be part or active players in the modulation of the store-operated calcium entry (SOCE). In this review we will analyze the evidence supporting macromolecular complex assembly as a prerequisite for SOC activation. We will highlight the evidence showing novel members from SOCIC and speculate about possible yet undiscovered members and players in this highly regulated calcium signaling mechanism. Finally we will discuss about the role of lipid raft domains in controlling store- and agonist-activated calcium influx.

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.

Visualizing the store-operated channel complex assembly in real time: Identification of SERCA2 as a new member

Depletion of intracellular calcium stores leads to the activation of calcium influx via the so-called store-operated channels (SOCs). Recent evidence positions Orai proteins as the putative channels responsible for this process. The stromal interacting molecule (STIM1) has been recently identified as the calcium sensor located at the endoplasmic reticulum (ER), and responsible for communicating the deplete state of calcium stores to Orai at the plasma membrane (PM). However, recent experimental findings suggest that Orai and STIM1 are only part of a larger molecular complex required to modulate store-operated calcium entry (SOCE). In the present study we describe the assembly of the several of the components from the SOC complex in real-time, utilizing a novel imaging method. Using FRET imaging we show that under resting conditions (with calcium stores replenished) STIM1 travels continuously through the ER associated to the microtubule tracking protein, EB1. Upon depletion of the ER STIM1 dissociates from EB1 and aggregates into macromolecular complexes at the ER which includes the microsomal calcium ATPase. This association follows the assembly of Orai into macromolecular aggregates at the PM. We show that STIM1-Orai association follows a similar time course as that of Orai aggregation at the PM. During this last step of the process, calcium-selective, whole-cell inward currents developed, simultaneously. We show that this process is fully reversible. Replenishing intracellular calcium stores induces STIM1-Orai complex dissociation and shuts down inward currents. Under these conditions STIM1 re-associates to EB1, and reinitiates its travel through the ER.

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization‐activated cyclic nucleotide‐gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately −20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about −20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately −50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.

Expression profiling of synaptic microRNAs from the adult rat brain identifies regional differences and seizure-induced dynamic modulation

In recent years, microRNAs or miRNAs have been proposed to target neuronal mRNAs localized near the synapse, exerting a pivotal role in modulating local protein synthesis, and presumably affecting adaptive mechanisms such as synaptic plasticity. In the present study we have characterized the distribution of miRNAs in five regions of the adult mammalian brain and compared the relative abundance between total fractions and purified synaptoneurosomes (SN), using three different methodologies. The results show selective enrichment or depletion of some miRNAs when comparing total versus SN fractions. These miRNAs were different for each brain region explored. Changes in distribution could not be attributed to simple diffusion or to a targeting sequence inside the miRNAs. In silico analysis suggest that the differences in distribution may be related to the preferential concentration of synaptically localized mRNA targeted by the miRNAs. These results favor a model of co-transport of the miRNA-mRNA complex to the synapse, although further studies are required to validate this hypothesis. Using an in vivo model for increasing excitatory activity in the cortex and the hippocampus indicates that the distribution of some miRNAs can be modulated by enhanced neuronal (epileptogenic) activity. All these results demonstrate the dynamic modulation in the local distribution of miRNAs from the adult brain, which may play key roles in controlling localized protein synthesis at the synapse.

Calmodulin Modulates the Delay Period between Release of Calcium from Internal Stores and Activation of Calcium Influx via Endogenous TRP1 Channels

In the present study we have explored the role of calmodulin (CaM) and inositol 1,4,5-trisphosphate receptor (IP3R) in the communication process activated after the release of calcium from the endoplasmic reticulum (ER) and the activation of calcium influx via endogenous TRP1 channels from Chinese hamster ovary cells. Experiments using combined rapid confocal calcium and electrophysiology measurements uncovered a consistent delay of around 900 ms between the first detectable calcium released from the ER and the activation of the calcium current. This delay was evident with two different methods used to release calcium from the ER: either the blockade of the microsomal calcium ATPase with thapsigargin or activation of bradykinin receptors linked to the IP3cascade. Direct application of IP3 or a peptide from the NH2-terminal region of the IP3R activated store operated calcium, reducing the delay period. Introduction of CaM into the cell via the patch pipette increased the delay period from 900 ± 100 ms to 10 ± 2.1 s (n = 18). Furthermore, the use of selective CaM antagonists W7 and trifluoperazine maleate resulted in a substantial reduction of the delay period to 200 ± 100 ms with 5 μmtrifluoperazine maleate (n = 16) and 150 ± 50 ms with 500 nm W7 (n = 22). CaM reduced also the current density activated by thapsigargin or brandykinin to about 60% from control. The CaM antagonists did not affect significantly the current density. The results presented here are consistent with an antagonistic effect of IP3R and CaM for the activation of store operated calcium after depletion of the ER. The functional competition between the activating effect of IP3R and the inhibiting effect of CaM may modulate the delay period between the release of calcium from the ER and the activation of calcium influx observed in different cells, as well as the amount of current activated after depletion of the ER.

Functional Reorganization of Visual Cortex Maps after Ischemic Lesions Is Accompanied by Changes in Expression of Cytoskeletal Proteins and NMDA and GABAA Receptor Subunits

Reorganization of cortical representations after focal visual cortex lesions has been documented. It has been suggested that functional reorganization may rely on cellular mechanisms involving modifications in the excitatory/inhibitory neurotransmission balance and on morphological changes of neurons peripheral to the lesion. We explored functional reorganization of cortical retinotopic maps after a focal ischemic lesion in primary visual cortex of kittens using optical imaging of intrinsic signals. After 1, 2, and 5 weeks postlesion (wPL), we addressed whether functional reorganization correlated in time with changes in the expression of MAP-2, GAP-43, GFAP, GABAA receptor subunit α1 (GABAAα1), subunit 1 of the NMDA receptor (NMDAR1), and in neurotransmitter levels at the border of the lesion. Our results show that: (1) retinotopic maps reorganize with time after an ischemic lesion; (2) MAP-2 levels increase gradually from 1wPL to 5wPL; (3) MAP-2 upregulation is associated with an increase in dendritic-like structures surrounding the lesion and a decrease in GFAP-positive cells; (4) GAP-43 levels reach the highest point at 2wPL; (5) NMDAR1 and glutamate contents increase in parallel from 1wPL to 5wPL; (6) GABAAα1 levels increase from 1wPL to 2wPL but do not change after this time point; and (7) GABA contents remain low from 1wPL to 5wPL. This is a comprehensive study showing for the first time that functional reorganization correlates in time with dendritic sprouting and with changes in the excitatory/inhibitory neurotransmission systems previously proposed to participate in cortical remodeling and suggests mechanisms by which plasticity of cortical representations may occur.

Osmotic swelling-induced changes in cytosolic calcium do not affect regulatory volume decrease in rat cultured suspended cerebellar astrocytes.

Abstract: Hyposmotic swelling‐induced changes in intracellular Ca2+ concentration ([Ca2+]i) and their influence on regulatory volume decrease (RVD) were examined in rat cultured suspended cerebellar astrocytes. Hyposmotic media (50 or 30%) evoked an immediate rise in [Ca2+]i from 117 nM to a mean peak increase of 386 (50%) and 220 nM (30%), followed by a maintained plateau phase. Ca2+ influx through the plasmalemma as well as release from internal stores contributed to this osmosensitive [Ca2+]i elevation. Omission of external Ca2+ or addition of Cd2+, Mn2+, or Gd3+ did not reduce RVD, although it was decreased by La3+ (0.1–1 mM). Verapamil did not affect either the swelling‐evoked [Ca2+]i or RVD. Maneuvers that deplete endoplasmic reticulum (ER) Ca2+ stores, such as treatment (in Ca2+‐free medium) with 0.2 µM thapsigargin (Tg), 10 µM 2,5‐di‐tert‐butylhydroquinone, 1 µM ionomycin, or 100 µM ATP abolished the increase in [Ca2+]i but did not affect RVD. However, prolonged exposure to 1 µM Tg blocked RVD regardless of ER Ca2+ content or cytosolic Ca2+ levels. Ryanodine (up to 100 µM) and caffeine (10 mM) did not modify [Ca2+]i or RVD. BAPTA‐acetoxymethyl ester (20 µM) abolished [Ca2+]i elevation without affecting RVD, but at higher concentrations BAPTA prevented cell swelling and blocked RVD. We conclude that the osmosensitive [Ca2+]i rise occurs as a consequence of increased Ca2+ permeability of plasma and organelle membranes, but it appears not relevant as a transduction signal for RVD in rat cultured cerebellar astrocytes.

Isovolumetric regulation mechanisms in cultured cerebellar granule neurons

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (− 1.8 mOsm/min) maintained constant volume up to − 50% external osmolarity (πo), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl−, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, d‐[3H]aspartate (as marker for glutamate) of πo− 2% and − 19%, respectively, and more delayed thresholds of − 30% for [3H]glycine and − 25% and − 29%, respectively, for Cl− (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five‐fold over other amino acids) and the largest cell content decrease. Taurine and Cl− efflux were abolished by niflumic acid and 86Rb by 15 mm Ba2+. Niflumic acid essentially prevented IVR in all ranges of πo. Cl−‐free medium impaired IVR when πo decreased to − 24% and Ba2+ blocked it only at a late phase of − 30% πo. These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of πo is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl− and amino acids, particularly taurine; (iii) Cl− contribution to IVR is delayed with respect to other niflumic acid‐sensitive osmolyte fluxes (osmolarity threshold of − 25% πo); and (iv), K+ fluxes do not contribute to IVR until a late phase (< − 30% πo).