All three IP3 receptor subtypes generate Ca2+ puffs, the universal building blocks of IP3-evoked Ca2+ signals

Abstract

All three subtypes of inositol 1,4,5-trisphosphate receptor (IP3R) are intracellular Ca2+ channels that are co-regulated by IP3 and Ca2+. This allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs that reflect the coordinated opening of a few clustered IP3Rs. We use total internal reflection microscopy (TIRF) microscopy to record Ca2+ signals in HEK cells expressing all three IP3R subtypes or a single native subtype. Ca2+ puffs are less frequent in cells expressing one IP3R subtype, commensurate with them expressing fewer IP3Rs than wild-type cells. However, all three IP3R subtypes generate broadly similar Ca2+ puffs with similar numbers of IP3Rs contributing to each. This suggests that IP3R clusters may be assembled by conserved mechanisms that generate similarly sized clusters across different IP3R expression levels. The Ca2+ puffs evoked by IP3R2 had slower kinetics and more prolonged durations, which may be due to IP3 binding with greater affinity to IP3R2. We conclude that Ca2+ puffs are the building blocks for the Ca2+ signals evoked by all IP3Rs.

IP3 Receptors Preferentially Associate with ER-Lysosome Contact Sites and Selectively Deliver Ca2+ to Lysosomes

“…The Ca 2+ signals evoked by histamine in HeLa cells or by CCh in HEK cells are initiated by IP 3 Rs, consistent with the lack of response to CCh in HEK cells without IP 3 Rs (Figure S1M). The HEK cells used express all three IP 3 R subtypes (IP 3 R3 > IP 3 R1 > IP 3 R2) (Mataragka and Taylor, 2018). We also used HEK cells where genes for one or two of the three IP 3 R subtypes were disrupted (Alzayady et al., 2016) to establish whether any IP 3 R subtype selectively presents Ca 2+ to lysosomes.…”

Ca2+ Release by IP3 Receptors Is Required to Orient the Mitotic Spindle

“…HEK cells express all three IP 3 R subtypes ( Mataragka and Taylor, 2018 ). In cells expressing only a single IP 3 R subtype, spindle angles were aberrant in cells expressing only IP 3 R1 (23.4° ± 14.7°, n = 72), but more normal in cells expressing only IP 3 R2 (18.1° ± 14.4°, n = 51) or IP 3 R3 (17.7° ± 13.9°, n = 66) ( Figures S2 D–S2F).…”

Structure and Function of IP3Receptors

“…There is also evidence that clustering may be required for cells to generate effective Ca 2+ signals (Geyer et al 2015). There is, however, a conundrum because, whereas most IP 3 Rs are mobile, the Ca 2+ puffs evoked by IP 3 repeatedly initiate at the same fixed sites within a cell (Thomas et al 1998;Smith and Parker 2009;Smith et al 2009b;Keebler and Taylor 2017;Mataragka and Taylor 2018). Why should a small fraction of the IP 3 Rs in a cell assume complete responsibility for generating Ca 2+ puffs?…”