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Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

López Sanjurjo CI, Tovey SC, Taylor CW - PLoS ONE (2014)

Bottom Line: The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1.Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release.We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

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Associations of ER with other Ca2+-sequestering organelles allows selective and reversible modulation of cytosolic Ca2+ signals.Close association of lysosomes (LY) with ER [30], probably mediated by specific tethers (red) [46], allows them selectively to accumulate Ca2+ released by IP3Rs from distinct ER Ca2+ stores, but not Ca2+ entering the cell via SOCE. Mitochondria (right), depending on cell type, can selectively accumulate Ca2+ released from the ER, to which they are tethered, or entering the cell via SOCE [11]. For lysosomes, neither the Ca2+ uptake pathway (1) nor the efflux pathway (2) that rapidly recycles Ca2+ back to the ER via the cytosol have been identified. The equivalent pathways in mitochondria are the MCU (1) and Na+/Ca2+ or H+/Ca2+ exchangers (2) of the inner mitochondrial membrane. Rapid, reversible and selective ‘buffering’ of cytosolic Ca2+ signals by both lysosomes and mitochondria allows these organelles to both shape and decode stimulus-evoked Ca2+ signals.
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pone-0111275-g006: Associations of ER with other Ca2+-sequestering organelles allows selective and reversible modulation of cytosolic Ca2+ signals.Close association of lysosomes (LY) with ER [30], probably mediated by specific tethers (red) [46], allows them selectively to accumulate Ca2+ released by IP3Rs from distinct ER Ca2+ stores, but not Ca2+ entering the cell via SOCE. Mitochondria (right), depending on cell type, can selectively accumulate Ca2+ released from the ER, to which they are tethered, or entering the cell via SOCE [11]. For lysosomes, neither the Ca2+ uptake pathway (1) nor the efflux pathway (2) that rapidly recycles Ca2+ back to the ER via the cytosol have been identified. The equivalent pathways in mitochondria are the MCU (1) and Na+/Ca2+ or H+/Ca2+ exchangers (2) of the inner mitochondrial membrane. Rapid, reversible and selective ‘buffering’ of cytosolic Ca2+ signals by both lysosomes and mitochondria allows these organelles to both shape and decode stimulus-evoked Ca2+ signals.

Mentions: We have shown that lysosomes sequester Ca2+ released from the ER [30]. The present work demonstrates that different IP3-sensitive Ca2+ stores within the compartmentalized ER of HEK cells [31] are each capable of directing the Ca2+ released by IP3Rs to lysosomal Ca2+ uptake systems (Figures 1 and 6). By contrast the Ca2+ signals evoked by SOCE, whether activated pharmacologically [30] or by endogenous receptors that stimulate PLC (Figure 4), are insensitive to inhibition of lysosomes. This is not due to the small amplitude of SOCE-mediated Ca2+ signals (Figures 3A and 4) because SOCE remains insensitive to inhibition of lysosomes when SOCE-evoked increases in global [Ca2+]i are larger than those evoked by IP3Rs [30]. The insensitivity of CCh-evoked SOCE to inhibition of lysosomal Ca2+ uptake suggests two important conclusions. First, it reinforces our suggestion that lysosomes selectively sequester Ca2+ released by IP3Rs [30]. The intimacy of the relationship between ER and lysosomes is further supported by the different effects of inhibiting lysosomes (Figure 1) or Ca2+ extrusion across the plasma membrane (Figure 5C). Only the former increases the amplitude of the initial CCh-evoked increase in [Ca2+]i, suggesting that only lysosomes are both close enough to IP3Rs and accumulate Ca2+ fast enough to attenuate the initial response to IP3. Second, it suggests that during SOCE in HEK cells, there is probably no significant flux of Ca2+ from Orai channels into the ER and then back into the cytosol via IP3Rs (Figure 4A lower panel).


Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

López Sanjurjo CI, Tovey SC, Taylor CW - PLoS ONE (2014)

Associations of ER with other Ca2+-sequestering organelles allows selective and reversible modulation of cytosolic Ca2+ signals.Close association of lysosomes (LY) with ER [30], probably mediated by specific tethers (red) [46], allows them selectively to accumulate Ca2+ released by IP3Rs from distinct ER Ca2+ stores, but not Ca2+ entering the cell via SOCE. Mitochondria (right), depending on cell type, can selectively accumulate Ca2+ released from the ER, to which they are tethered, or entering the cell via SOCE [11]. For lysosomes, neither the Ca2+ uptake pathway (1) nor the efflux pathway (2) that rapidly recycles Ca2+ back to the ER via the cytosol have been identified. The equivalent pathways in mitochondria are the MCU (1) and Na+/Ca2+ or H+/Ca2+ exchangers (2) of the inner mitochondrial membrane. Rapid, reversible and selective ‘buffering’ of cytosolic Ca2+ signals by both lysosomes and mitochondria allows these organelles to both shape and decode stimulus-evoked Ca2+ signals.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206489&req=5

pone-0111275-g006: Associations of ER with other Ca2+-sequestering organelles allows selective and reversible modulation of cytosolic Ca2+ signals.Close association of lysosomes (LY) with ER [30], probably mediated by specific tethers (red) [46], allows them selectively to accumulate Ca2+ released by IP3Rs from distinct ER Ca2+ stores, but not Ca2+ entering the cell via SOCE. Mitochondria (right), depending on cell type, can selectively accumulate Ca2+ released from the ER, to which they are tethered, or entering the cell via SOCE [11]. For lysosomes, neither the Ca2+ uptake pathway (1) nor the efflux pathway (2) that rapidly recycles Ca2+ back to the ER via the cytosol have been identified. The equivalent pathways in mitochondria are the MCU (1) and Na+/Ca2+ or H+/Ca2+ exchangers (2) of the inner mitochondrial membrane. Rapid, reversible and selective ‘buffering’ of cytosolic Ca2+ signals by both lysosomes and mitochondria allows these organelles to both shape and decode stimulus-evoked Ca2+ signals.
Mentions: We have shown that lysosomes sequester Ca2+ released from the ER [30]. The present work demonstrates that different IP3-sensitive Ca2+ stores within the compartmentalized ER of HEK cells [31] are each capable of directing the Ca2+ released by IP3Rs to lysosomal Ca2+ uptake systems (Figures 1 and 6). By contrast the Ca2+ signals evoked by SOCE, whether activated pharmacologically [30] or by endogenous receptors that stimulate PLC (Figure 4), are insensitive to inhibition of lysosomes. This is not due to the small amplitude of SOCE-mediated Ca2+ signals (Figures 3A and 4) because SOCE remains insensitive to inhibition of lysosomes when SOCE-evoked increases in global [Ca2+]i are larger than those evoked by IP3Rs [30]. The insensitivity of CCh-evoked SOCE to inhibition of lysosomal Ca2+ uptake suggests two important conclusions. First, it reinforces our suggestion that lysosomes selectively sequester Ca2+ released by IP3Rs [30]. The intimacy of the relationship between ER and lysosomes is further supported by the different effects of inhibiting lysosomes (Figure 1) or Ca2+ extrusion across the plasma membrane (Figure 5C). Only the former increases the amplitude of the initial CCh-evoked increase in [Ca2+]i, suggesting that only lysosomes are both close enough to IP3Rs and accumulate Ca2+ fast enough to attenuate the initial response to IP3. Second, it suggests that during SOCE in HEK cells, there is probably no significant flux of Ca2+ from Orai channels into the ER and then back into the cytosol via IP3Rs (Figure 4A lower panel).

Bottom Line: The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1.Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release.We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

Show MeSH
Related in: MedlinePlus