Overexpression of certain transient receptor potential and Orai channels in prostate cancer is associated with decreased risk of systemic recurrence after radical prostatectomy.

BACKGROUND: Several studies had suggested the potential role of calcium signaling in prostate cancer (PCa) prognosis and agressiveness. We aimed to investigate selected proteins contributing to calcium (Ca2+ ) signaling, (Orai, stromal interaction molecule (STIM), and transient receptor potential (TRP) channels) and involved in cancer hallmarks, as independent predictors of systemic recurrence after radical prostatectomy (RP). METHODS: A case-control study including 112 patients with clinically localized PCa treated by RP between 2002 and 2009 and with at least 6-years' follow-up. Patients were divided into two groups according to the absence or presence of systemic recurrence. Expression levels of 10 proteins involved in Ca2+ signaling (TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, STIM1, STIM2, Orai1, Orai2, and Orai3), were assessed by immunohistochemistry using tissue microarrays (TMAs) constructed from paraffin-embedded PCa specimens. The level of expression of the various transcripts in PCa was assessed using quantitative polymerase chain reaction (qPCR) analysis. RNA samples for qPCR were obtained from fresh frozen tissue samples of PCa after laser capture microdissection on RP specimens. Relative gene expression was analyzed using the 2-▵▵Ct method. RESULTS: Multivariate analysis showed that increased expression of TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, and Orai2 was significantly associated with a lower risk of systemic recurrence after RP, independently of the prostate-specific antigen (PSA) level, percentage of positive biopsies, and surgical margin (SM) status (P = .007, P = .01, P < .001, P = .0065, P = .007, and P = .01, respectively). For TRPC4, TRPV5, and TRPV6, this association was also independent of Gleason score and pT stage. Moreover, overexpression of TRPV6 and Orai2 was significantly associated with longer time to recurrence after RP (P = .048 and .023, respectively). Overexpression of TRPC4, TRPV5, TRPV6, and Orai2 transcripts was observed in group R- (3.71-, 5.7-, 1.14-, and 2.65-fold increase, respectively). CONCLUSIONS: This is the first study to suggest the independent prognostic value of certain proteins involved in Ca2+ influx in systemic recurrence after RP: overexpression of TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, and Orai2 is associated with a lower risk of systemic recurrence. TRPC4, TRPV5, and TRPV6 appear to be particularly interesting, as they are independent of the five commonly used predictive factors, that is, PSA, percentage of positive biopsies, SM status, Gleason score, and pT stage.

The SigmaR1 chaperone drives breast and colorectal cancer cell migration by tuning SK3-dependent Ca2+ homeostasis.

The remodeling of calcium homeostasis contributes to the cancer hallmarks and the molecular mechanisms involved in calcium channel regulation in tumors remain to be characterized. Here, we report that SigmaR1, a stress-activated chaperone, is required to increase calcium influx by triggering the coupling between SK3, a Ca2+-activated K+ channel (KCNN3) and the voltage-independent calcium channel Orai1. We show that SigmaR1 physically binds SK3 in BC cells. Inhibition of SigmaR1 activity, either by molecular silencing or by the use of sigma ligand (igmesine), decreased SK3 current and Ca2+ entry in breast cancer (BC) and colorectal cancer (CRC) cells. Interestingly, SigmaR1 inhibition diminished SK3 and/or Orai1 levels in lipid nanodomains isolated from BC cells. Analyses of tissue microarray from CRC patients showed higher SigmaR1 expression levels in cancer samples and a correlation with tumor grade. Moreover, the exploration of a cohort of 4937 BC patients indicated that high expression of SigmaR1 and Orai1 channels was significantly correlated to a lower overall survival. As the SK3/Orai1 tandem drives invasive process in CRC and bone metastasis progression in BC, our results may inaugurate innovative therapeutic approaches targeting SigmaR1 to control the remodeling of Ca2+ homeostasis in epithelial cancers.

Function and regulation of TRPM7, as well as intracellular magnesium content, are altered in cells expressing ΔF508-CFTR and G551D-CFTR.

Cystic fibrosis (CF), one of the most common fatal hereditary disorders, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene product is a multidomain adenosine triphosphate-binding cassette (ABC) protein that functions as a chloride (Cl(-)) channel that is regulated by intracellular magnesium [Mg(2+)]i. The most common mutations in CFTR are a deletion of a phenylalanine residue at position 508 (ΔF508-CFTR, 70-80 % of CF phenotypes) and a Gly551Asp substitution (G551D-CFTR, 4-5 % of alleles), which lead to decreased or almost abolished Cl(-) channel function, respectively. Magnesium ions have to be finely regulated within cells for optimal expression and function of CFTR. Therefore, the melastatin-like transient receptor potential cation channel, subfamily M, member 7 (TRPM7), which is responsible for Mg(2+) entry, was studies and [Mg(2+)]i measured in cells stably expressing wildtype CFTR, and two mutant proteins (ΔF508-CFTR and G551D-CFTR). This study shows for the first time that [Mg(2+)]i is decreased in cells expressing ΔF508-CFTR and G551D-CFTR mutated proteins. It was also observed that the expression of the TRPM7 protein is increased; however, membrane localization was altered for both ΔF508del-CFTR and G551D-CFTR. Furthermore, both the function and regulation of the TRPM7 channel regarding Mg(2+) is decreased in the cells expressing the mutated CFTR. Ca(2+) influx via TRPM7 were also modified in cells expressing a mutated CFTR. Therefore, there appears to be a direct involvement of TRPM7 in CF physiopathology. Finally, we propose that the TRPM7 activator Naltriben is a new potentiator for G551D-CFTR as the function of this mutant increases upon activation of TRPM7 by Naltriben.

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells.

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca(2+)) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca(2+) signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca(2+)-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response.

Calcium entry and the control of calcium oscillations.

During oscillatory Ca(2+) signals, the agonist-induced enhanced entry of extracellular Ca(2+) plays a critical role in modulating the frequency of the oscillations. Although it was originally assumed that the entry of Ca(2+) under these conditions occurred via the well-known, and apparently ubiquitous, store-operated mechanism, subsequent studies suggested that this was unlikely. It is now known that, in many cell types, a novel non-capacitative Ca(2+)-selective pathway whose activation is dependent on arachidonic acid is responsible, and the channels involved [ARC channels (arachidonate-regulated Ca(2+) channels)] have been characterized. These ARC channels co-exist with the store-operated CRAC channels (Ca(2+)-release-activated Ca(2+) channel) in cells, but each plays a unique and non-overlapping role in Ca(2+) signalling. In particular, it is the ARC channels that are specifically activated at the low agonist concentrations that give rise to oscillatory Ca(2+) signals and provide the predominant mode of Ca(2+) entry under these conditions. The indications are that Ca(2+) entry through the ARC channels increases the likelihood that low concentrations of Ins(1,4,5)P(3) will trigger repetitive Ca(2+) release. At higher agonist concentrations, store-depletion is more complete and sustained resulting in the activation of CRAC channels. At the same time the ARC channels are turned off, resulting in what we have described as a reciprocal regulation of these two distinct Ca(2+) entry pathways.

Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca2+ entry pathways.

Receptor-activated Ca(2+) entry is usually thought to occur via capacitative or store-operated Ca(2+) channels. However, at physiological levels of stimulation, where Ca(2+) store depletion is only transient and/or partial, evidence has suggested that an arachidonic acid-dependent noncapacitative Ca(2+) entry is responsible. Recently, we have described a novel arachidonate-regulated Ca(2+)-selective (ARC) conductance that is entirely distinct from store-operated conductances in the same cell. We now show that these ARC channels are indeed specifically activated by low agonist concentrations and provide the predominant route of Ca(2+) entry under these conditions. We further demonstrate that sustained elevations in cytosolic Ca(2+), such as those resulting from activation of store-operated Ca(2+) entry by high agonist concentrations, inhibit the ARC channels. This explains earlier failures to detect the presence of this noncapacitative pathway in experiments where store-operated entry had already been fully activated. The result is that the respective activities of ARC and store-operated Ca(2+) channels display a unique reciprocal regulation that is related to the specific nature of the [Ca(2+)](i) signals generated at different agonist concentrations. Importantly, these data show that at physiologically relevant levels of stimulation, it is the noncapacitative ARC channels that provide the predominant route for the agonist-activated entry of Ca(2+).

Permeation of monovalent cations through the non-capacitative arachidonate-regulated Ca2+ channels in HEK293 cells. Comparison with endogenous store-operated channels.

In a manner similar to voltage-gated Ca(2+) channels and Ca(2+) release-activated Ca(2+) (CRAC) channels, the recently identified arachidonate-regulated Ca(2+) (ARC) channels display a large monovalent conductance upon removal of external divalent cations. Using whole-cell patch-clamp recording, we have characterized the properties of these monovalent currents in HEK293 cells stably transfected with the m3 muscarinic receptor and compared them with the corresponding currents through the endogenous store-operated Ca(2+) (SOC) channels in the same cells. Although the monovalent currents seen through these two channels displayed certain similarities, several marked differences were also apparent, including the magnitude of the monovalent current/Ca(2+) current ratio, the rate and nature of the spontaneous decline in the currents, and the effects of external monovalent cation substitutions and removal of internal Mg(2+). Moreover, monovalent ARC currents could be activated after the complete spontaneous inactivation of the corresponding SOC current in the same cell. We conclude that the non-capacitative ARC channels share, with voltage-gated Ca(2+) channels and store-operated Ca(2+) channels (e.g. SOC and CRAC the general property of monovalent ion permeation in the nominal absence of extracellular divalent ions. However, the clear differences between the properties of these currents through ARC and SOC channels in the same cell confirm that these represent distinct conductances.

Basolateral outward rectifier chloride channel in isolated crypts of mouse colon.

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between -100 and -40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I(-) > SCN(-) > Br(-) > Cl(-) > NO(3)(-) > F(-)>> SO(4)(2-) approximately gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca(2+) concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5'-O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5'-O-(2-thiodiphosphate) reactivated the channel.

I(ARC), a novel arachidonate-regulated, noncapacitative Ca(2+) entry channel.

Along with the inositol trisphosphate-induced release of stored Ca(2+), a receptor-enhanced entry of Ca(2+) is a critical component of intracellular Ca(2+) signals generated by agonists acting at receptors coupled to the activation of phospholipase C. Although the simple emptying of the intracellular Ca(2+) stores is known to be capable of activating Ca(2+) entry via the so-called "capacitative" mechanism, recent evidence suggests that Ca(2+) entry at physiological agonist concentrations, where oscillatory Ca(2+) signals are typically observed, does not conform to such a model. Instead, a noncapacitative Ca(2+) entry pathway regulated by arachidonic acid appears to be responsible for Ca(2+) entry under these conditions. Using whole-cell patch clamp techniques we demonstrate that low concentrations of arachidonic acid activate a Ca(2+)-selective current that is superficially similar to the store-operated current I(CRAC), but which also demonstrates certain distinct features. We have named this novel current I(ARC) (for arachidonate-regulated calcium current). Importantly, I(ARC) can be readily activated in cells whose Ca(2+) stores have been maximally depleted. I(ARC) represents a novel Ca(2+) entry pathway that is entirely separate from those activated by store depletion and is specifically activated at physiological levels of stimulation.

Volume regulation following hypotonic shock in isolated crypts of mouse distal colon.

1. A video-imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2. Transition from isotonic (310 mosmol l-1) to hypotonic (240 mosmol l-1) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3. Exposure of crypts to Cl--free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na+-free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4. The chloride channel inhibitor NPPB (50 microM) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5. The K+ channel blockers barium (10 mM), charybdotoxin (10 nM) and TEA (5 mM) inhibited RVD by 51, 44 and 32 %, respectively. 6. Intracellular [Ca2+] rose from a baseline of 174 +/- 17 nM (n = 8) to 448 +/- 45 nM (n = 8) during the initial swelling phase 7. The Ca2+ channel blockers verapamil (50 microM) and nifedipine (10 microM), the chelator of intracellular Ca2+ BAPTA AM (30 microM), or the inhibitor of Ca2+ release TMB-8 (10 microM), dramatically reduced volume recovery, leading to 51 % (n = 9), 25 % (n = 7), 37 % (n = 6), 32 % (n = 8) inhibition of RVD, respectively. TFP (50 microM), an antagonist of the Ca2+-calmodulin complex, significantly slowed RVD. The Ca2+ ionophore A23187 (2 microM) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca2+ from intracellular stores using bradykinin (1 microM) or blockade of reabsorption with thapsigargin (1 microM) decreased the duration of RVD. 8. Prostaglandin E2 (PGE2, 5 microM) slightly delayed RVD, whereas leukotriene D4 (LTD4, 100 nM) and arachidonic acid (10 microM) reduced the duration of RVD. Blockade of phospholipase A2 by quinacrine (10 microM) inhibited RVD by 53 %. Common inhibition of PGE2 and LTD4 synthesis by ETYA (50 microM) or separate blockade of PGE2 synthesis by 1 microM indomethacin reduced the duration of RVD. Blockade of LTD4 synthesis by nordihydroguaiaretic acid (NDGA) did not produce any significant effect on cell swelling or subsequent RVD. 9. Staurosporine (1 microM), an inhibitor of protein kinases, inhibited RVD by 58 %. Taken together the experiments demonstrate that the RVD process is under the control of conductive pathways, extra- and intracellular Ca2+ ions, protein kinases, prostaglandins and leukotrienes.

Chloride and non-selective cation channels in unstimulated trout red blood cells.

1. The cell-attached and excised inside-out configurations of the patch-clamp technique were used to demonstrate the presence of two different types of ion channels in the membrane of trout red blood cells under isotonic and normoxic conditions, in the absence of hormonal stimulation. The large majority (93%) of successful membrane seals allowed observation of at least one channel type. 2. In the cell-attached mode with Ringer solution in the bath and Ringer solution, 145 mM KCl or 145 NaCl in the pipette, a channel of intermediate conductance (15-25 pS at clamped voltage, Vp = 0 mV) was present in 85% of cells. The single channel activity reversed between 5 and 7 mV positive to the spontaneous membrane potential. A small conductance channel of 5-6 pS and +5 mV reversal potential was also present in 62% of cells. 3. After excision into the inside-out configuration (with 145 mM KCl or NaCl, pCa 8 in the bath, 145 mM KCl or NaCl, pCa 3 in the pipette) the intermediate conductance channel was present in 439 out of 452 successful seals. This channel was spontaneously active in 90% of patches and in the other 10% of patches the channel was activated by suction. The current-voltage relationship showed slight inward rectification. The channel conductance was in the range 15-20 pS between -60 and 0 mV and increased to 25-30 pS between 0 and 60 mV, with a reversal potential close to zero. Substitution of K+ for Na+ in the pipette or in the bath did not significantly change the single channel conductance. Dilution of the bathing solution KCl concentration shifted the reversal potential towards the Nernst equilibrium for cations. Substitution of N-methyl-D-glucamine (NMDG) for K+ or Na+ in the bath almost abolished the outward current whilst the divalent cation Ca2+ permeated the channel with a higher permeability than K+ and Na+. Inhibition of channel openings was obtained with flufenamic acid, quinine, gadolinium or barium. Taken together these data demonstrate that the intermediate conductance channel belongs to a class of non-selective cation (NSC) channels. 4. In excised patches, under the same control conditions, the conductance of the small conductance non-rectifying channel was 8.6 +/- 0.8 pS (n = 12) between -60 and +60 mV and the reversal potential was close to 0 mV. This channel could be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) but not by flufenamic acid, DIDS, barium or gadolinium. Selectivity and substitution experiments made it possible to identify this channel as a non-rectifying small conductance chloride (SCC) channel.