Abstract
ABSTRACTAll 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.
Highlights
Many receptors evoke Ca2+ signals by stimulating phospholipase C (Berridge, 2016)
By using an antibody that recognises a sequence conserved in all IP3 receptors (IP3Rs) subtypes (AbC), we established that overall IP3R expression levels were similar in each cell line expressing a single IP3R subtype (Fig. 1D), but lower (∼32%) than in WT cells (Fig. 1D)
IP3Rs is anchored near the plasma membrane, where an additional signal licenses the cluster to respond to IP3 (Smith et al, 2009a; Thillaiappan et al, 2017)
Summary
Many receptors evoke Ca2+ signals by stimulating phospholipase C (Berridge, 2016). Inositol 1,4,5-trisphosphate (IP3) evokes Ca2+ release from the endoplasmic reticulum by stimulating IP3 receptors (IP3Rs), which are widely expressed Ca2+ channels. IP3 binding to IP3R initiates its activation by promoting the Ca2+ binding that leads to channel opening (Prole and Taylor, 2016) This dual regulation allows IP3Rs to evoke regenerative Ca2+ signals, the smallest of which are Ca2+ puffs (Smith and Parker, 2009). These arise from the nearly simultaneous opening of a few clustered IP3Rs as Ca2+ released by one IP3R ignites the activity of its neighbours. Ca2+ puffs are thought to be fundamental building blocks of IP3evoked Ca2+ signalling They require assembly of IP3Rs into small clusters and their anchoring at sites close to the plasma membrane (Smith et al, 2009a) where additional signals prime or ‘license’ IP3Rs to respond (Thillaiappan et al, 2017)
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