Free calcium in the cytosol is critical for regulating cellular processes in response to intracellular and extracellular stimuli. Calcium signaling involves the opening of calcium channels within the plasma membrane or membranes of intracellular organelles, most notably the endoplasmic reticulum (ER). Extracellular stimuli lead to the release of calcium from the ER in response to calcium or inositol 1,4,5-triphosphate (IP3) binding the IP3 receptors (IP3Rs).
The location of calcium release into the cytosol elicits different responses. Lysosomes can dichotomously influence calcium release through multiple channels – transient receptor potential mucolipin (TRPML), two-pore channel 2 (TPC2), ATP-regulated P2X4 receptors – as well as sequester calcium release. Contact between lysosomes and the ER via membrane contact sites (MCS) is known to be involved in regulating calcium release and calcium sequestering. Sequestering calcium release from the ER attenuates cytosolic calcium signals by IP3Rs, a process that remains unclear.
Researchers at the University of Cambridge sought to study the mechanism by which lysosomes sequester calcium release from the ER, publishing their findings in Cell Reports. We were excited to see our IP3R Expressing HEK-293 Cell Lines, developed by the laboratory of David I. Yule, PhD, at University of Rochester, cited in the paper.
Luminal lysosome pH is maintained by H+-ATPase (V-ATPase). HEK cells were treated with a selective inhibitor of V-ATPase, concanamycin A (CcA), to disrupt the pH gradient leading to a mild increase in calcium levels. Treatment of the cells with carbochol (CCh), a compound that increases free calcium by stimulating IP3 formation, and CcA led to a significant increase in calcium. Similar results were reported when the V-ATPase was disrupted with siRNA, indicating that CCh-induced increases in calcium are dependent on inhibiting V-ATPase. Further experiments demonstrated that store-operated calcium entry (SOCE) was not affected by V-ATPase inhibition, signifying an important role for the lysosomal pH gradient in regulating cytosolic calcium increases by IP3Rs.
Low-affinity calcium sensors targeted to the cytosolic surface of lysosomes were used to detect local changes in calcium in HeLa cells that are stimulated by histamine to induce IP3 formation and calcium release from the ER. IP3Rs were found to deliver calcium to 60% of lysosomes, which can occur through all three receptor subtypes. MCS were found to facilitate the transfer of calcium between the ER and organelles, including lysosomes. Contact is maintained between lysosomes and the ER despite the organelles moving around and persistent contact was observed in regions with clusters of IP3Rs. Disruption of the MCS caused by inhibition of V-ATPase is believed to be preventing lysosomes from sequestering calcium.
If you study calcium and IP3Rs, check out our IP3R expressing HEK-293 cell lines here. You might also be interested in several other reagents for calcium signaling research:
- RyR1 Expressing HEK293 Cell Line from University of Rochester
- Low Voltage-Activated, T-type, Ca+ Channel (Cav3) HEK293 Cell Lines from University of Virginia
- Connexin-32 Antibody from Harvard University
- Calcium Indicator GCaMP3 (CMV) Lentivirus