Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

Abstract

The inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is an intracellular Ca 2+ release channel, and its opening is controlled by IP 3 and Ca 2+ . A single IP 3 binding site and multiple Ca 2+ binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca 2+ is unknown. In this study, we visualized conformational changes in IP 3 R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP 3 and Ca 2+ have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca 2+ dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca 2+ alone from the FRET changes evoked by both ligands together. A mutant IP 3 R containing a single amino acid substitution at K508, which is critical for IP 3 binding, did not exhibit this bell-shaped Ca 2+ dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca 2+ sensor, resulted in w10-fold reduction in the Ca 2+ dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP 3 R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca 2+ binding affinity, as the mechanism underlying the IP 3 -dependent regulation of the bell-shaped relationship between the IP 3 R activity and cytosolic Ca 2+ .calcium signal | channel gating | ion channel T he inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R) is a dualligand-gated Ca 2+ release channel whose opening is controlled by IP 3 and Ca 2+ (1) and which plays a crucial role in the generation of Ca 2+ signals that control numerous cellular processes (2). Individual IP 3 R subunits possess a single IP 3 binding site (3) and multiple Ca 2+ binding sites (4, 5), and tetrameric complexes of these subunits form functional IP 3 -gated Ca 2+ release channels (6). Cytoplasmic Ca 2+ regulates IP 3 R in a biphasic manner: Ca 2+ release is potentiated at low Ca 2+ concentrations but inhibited at higher Ca 2+ concentrations (7,8). The stimulatory effect suggests that the channels display the process of Ca 2+ -induced Ca 2+ release, which underlies Ca 2+ spike generation and wave propagation. In other words, the bell-shaped dependence on cytosolic Ca 2+ is the fundamental property of IP 3 R for the generation of Ca 2+ excitability (9).IP 3 monotonically activates the IP 3 R channels at constant Ca 2+ concentrations (10), but IP 3 dynamically changes the Ca 2+ sensitivity of the channel (11,12). At subsaturating concentrations of IP 3 , the optimal Ca 2+ concentration for IP 3 R modulation becomes lower, whereas at very high concentrations of IP 3 , channel activity persists at supramicromolar Ca 2+ concentrations (11,12). This mechanism of dual-ligand regulat...

N-terminus oligomerization is conserved in intracellular calcium release channels

“…Moreover, two very recent reports concur with our present study, providing evidence for IP 3 R1 N-terminus oligomerization from bead aggregation and chemical cross-linking assays [20,21]. Indirect evidence is also provided by FRET measurements of two fluorescent proteins fused to the N-terminus of individual IP 3 R1 subunits, suggesting that the N-termini from adjacent subunits are in physical proximity [22]. Importantly, we have observed that IP 3 R N-terminus tetramerization appears to be independent of IP 3 at micromolar levels (Figures 6 and 7).…”

Inositol 1,4,5‐trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi

“…ER luminal Ca 2+ imaging of HeLa cells and epimastigotes was carried out as described (Shinohara et al ., ), with some modifications. Details are described in the figure legends and Supporting information .…”

Computational quest for understanding the role of astrocyte signaling in synaptic transmission and plasticity

“…IP 3 -triggered Ca 2+ -induced Ca 2+ release (CICR) from the ER is considered the primary mechanism responsible for intracellular Ca 2+ dynamics in astrocytes (Volterra and Meldolesi, 2005; Nimmerjahn, 2009). This mechanism, schematized in Figure 2A, is essentially controlled by the interplay of three fluxes: (1) a Ca 2+ transfer from the cytosol to the ER ( J P ) mediated by endoplasmic-reticulum Ca 2+ -ATPase (SERCA) pumps which contributes to the maintenance of higher Ca 2+ concentrations in the ER stores than in the cytosol; (2) a passive Ca 2+ leak ( J L ) from the ER to the cytosol that is driven by the Ca 2+ gradient between the ER and the cytosol; and (3) an efflux ( J NL ) from the ER to the cytosol through IP 3 receptor (IP 3 R) channels, which depends both on IP 3 and Ca 2+ concentrations in the cytosol in a nonlinear fashion (Bezprozvanny et al, 1991; Ramos-Franco et al, 2000; Shinohara et al, 2011). …”