Second Messenger oparated Ca+2 influx/SOCE

Application of 30µM ATP to a megakaryocyte results in an inward current with two phases: a large, transient influx through P2X and a smaller more sustained influx, mediated by P2Y. This is known as P2X -/- platelets still show the second component to the current.

Reverse transcription PCR of mRNA from megakaryocytes indicates that TRPC1 and TRPC6 are expressed in the cell. Only TRPC6 is activated by DAG so this is the only real candidate for mediating this current. Also, TRPC1 KO mice exhibit normal platelet responses.

Controversy surrounds this topic in the literature: some papers have suggested TRPC6 to play a role in thrombogenesis and haemostasis, whereas another paper has said TRPC6 -/- platelets are normal. A paper from last year suggested TRP channels on platelets function as coincidence detectors to mediate phosphatidylserine exposure. It has been proposed that Na+ influx through TRPC3 and 6 causes reversal of the sodium/calcium exchanger (NCX) to promote Ca2+ entry.

IP3 receptors are non-elective cation channels located in the ER  and are activated by cytosolic IP3/Ca2+, . In 2006, a paper provided evidence that there MAY be a PM-bound IP3R. This was thought as application of IP3 or an analog resulted in Ca2+ influx through the PM (this is not actually the case).

Yang et al. (2012) proposed TMEM16F to be cation-selective, allowing Ca2+ entry, which would pose another Ca2+ entry mechanism in these cells. However, Mahaut-Smith’s lab have been unable to replicate the Ca2+ current through TMEM16F. It is worth noting Yang et al.’s (2012) findings were under very high [Ca2+]o in excised patches. Addition to intracellular Ca2+ results in an outward current proportional to the amount inserted. Current recordings show strong outward rectification with reversal ~0mV - which does not correspond to TMEM16F -/-.

 The role of TMEM16F is still unclear. It may be that: Ca2+ entry through it may activate a scramblase, it may be a Ca2+-activated mediator of scrambles activity, or it may perform both functions of allowing Ca2+ entry and scrambling phospholipids. Yang et al.’s (2012) study suggests TMEM16F is important but not solely responsible for Ca2+-mediated scrambling for phosphatidyserine exposure (it still happens in TMEM16F KO).

The rise in [Ca2+] following PLCß/y activation by GPCRs or RTKs respectively is much reduced in the absence of extracellular calcium, indicating that store-operated calcium entry occurs following store depletion via IP3R activation by the GPCRs. Thapsigargin (SERCA inhibitor) and ionomycin (Ca2+ ionophore causing leak from ER) also cause Ca2+ influx through the PM.

The role of stromal interaction molecule 1 (STIM1) in SOCE was discovered in 2005. STIM1 activates the "store-operated" ORAI1 calcium ion channels in the plasma membrane, via intracellular STIM1 movement. STIM1 is a single TM protein with an EF Ca2+-sensing hand inside the ER which senses Ca2+ depletion. 

dOrai has three human homologs: Orai1, 2 and 3. Orai1 channel is 4TM with intracellular N and C termini. Immunohistochemistry illustrated that the protein is localised to the PM. The protein ORAI1 is a structural component of the Calcium release activated  calcium channel (CRAC). The calcium release-activated calcium (CRAC) channel was characterised in 1992. It strongly inwardly rectifying with a very high reversal potential unachievable under physiological conditions. It is highly Ca2+-selective and has a very low single-channel conductance.

The Ca2+-activated transcription factor NFAT (tagged with GFP) was used in a genome-wide interference study of Drosophila which possesses 21,000 genes. Knocking down dStim (Drosophila STIM) or dOrai prevented the Ca2+-induced NFAT translocation 10 minutes following TG application, implying they are key in SOCE. 

In order to find out how Stim1 activates ORAI1, total internal reflection fluorescence (TIRF) microscopy was used. Wide-field fluorescence microscopy (normal) using fluorescent Stim1 indicated that the protein translocates to the PM following store depletion. TIRF microscopy added to this by showing that Stim1 moves to discrete ‘puncti’. Image analysis showed Stim1 and ORAI1 to co-localise following store depletion. This was supported by FRET analysis indicating a proximity of 3-10nm. Furthermore, kinetics experiments showed that CRAC activation has a hill coefficient of 4.2, and Stim1 displays a close value of 3.8. This indicates high cooperativity.

In support of the theory for CRAC underlying SOCE entry in platelets, the Ca2+ response of Stim1 -/- platelets following the application of various agonists (ADP, thrombin, collage etc.) was recorded and found to be diminished.

Ca2+ influx into the immune cells of SCID patients following TG application showed that SOCE is abolished. WT cells show a significant Ca2+ influx. Parents of SCID patients show an intermediate phenotype, indicating a gene dosage effect takes place.Linkage analysis to identify SNPs which may contribute to SCID identified a 74 gene region which contains Orai1, with a probability ratio of 500,000:1. The Orai1 mutation was R91W.The Ca2+ current in an R91W mutant following store depletion is abolished as in SCID patient cells. Orai1 rescue (added back in) cells display WT CRAC currents, and re-introducing the R91W mutant gene does not.

Store depletion by ionomycin at high Ca2+ buffering in the megakaryocyte results in the production of an I-V curve like that of CRAC currents seen in lymphocytes (inwardly rectifying, non-reversing). Application of ADP under low Ca2+ buffering produces an outwardly rectifying curve like that seen for TRPC6. Application of ADP under high Ca2+ buffering produced a curve intermediate of these two, implying contribution by both channels. 

In conclusion, it is apparent that Orai1 is the primary channel underlying SOCE in platelets. As Orai1 -/- display reduced thrombus formation and infarct following ischaemia, without inducing excessive bleeding, the channel may present a good therapeutic target for anti-thrombotics. However, it appears TRPC6 may play a role in Ca2+ entry - it may be that the channel couples to Orai1, though it is unknown now.