Clustering of InsP3 receptors by InsP3 retunes their regulation by InsP3 and Ca2+

Abstract

The versatility of Ca2+ signals derives from their spatio-temporal organization. For Ca2+ signals initiated by inositol-1,4,5-trisphosphate (InsP3), this requires local interactions between InsP3 receptors (InsP3Rs) mediated by their rapid stimulation and slower inhibition\ by cytosolic Ca2+. This allows hierarchical recruitment of Ca2+ release events as the InsP3 concentration increases. Single InsP3Rs respond first, then clustered InsP3Rs open together giving a local 'Ca2+ puff', and as puffs become more frequent they ignite regenerative Ca2+ waves. Using nuclear patch-clamp recording, here we demonstrate that InsP3Rs are initially randomly distributed with an estimated separation of 1 m. Low concentrations of InsP3 cause InsP3Rs to aggregate rapidly and reversibly into small clusters of about four closely associated InsP3Rs. At resting cytosolic [Ca2+], clustered InsP3Rs open independently, but with lower open probability, shorter open time, and less InsP3 sensitivity than lone InsP3Rs. Increasing cytosolic [Ca2+] reverses the inhibition caused by clustering, InsP3R gating becomes coupled, and the duration of multiple openings is prolonged. Clustering both exposes InsP3Rs to local Ca2+ rises and increases the effects of Ca2+. Dynamic regulation of clustering by InsP3 retunes InsP3R sensitivity to InsP3 and Ca2+, facilitating hierarchical recruitment of the elementary events that underlie all InsP3-evoked Ca2+ signals.

Figure 1. IP₃R are randomly distributed

a, IP₃-evoked Ca²⁺ release from permeabilized DT40-IP₃R3 (EC₅₀ = 281 ± 46 nM) and DT40-KO cells (means ± SEM, n ≥ 3). Immunoblot with IP₃R3-specific antiserum (10 μg membrane protein/lane, 220-kDa marker shown). b, Currents recorded from excised patches with 10 μM IP₃ in PS. No currents were detected without IP₃ (n = 20), with heparin (100 μg/ml) and IP₃ (n = 15), or with IP₃ in DT40-KO cells (n > 30). C denotes closed state. c, i-V relationship for IP₃-evoked current (γ_K = 121 ± 2.8 pS, n = 7). d, Dwell time distribution of single IP₃R3 stimulated with 10 μM IP₃. Open time distribution of this typical recording is fitted with a single probability density function (pdf) with τ_o = 10.4 ms (mean = 11.9 ± 1.6 ms, n = 6). The pdf for the τ_c distribution has two components (1.07 ms, 88% and 109 ms, 12%). Dwell time distributions are consistent with the gating scheme (Supplementary Methods, Supplementary Figs 5, 6). e, Typical all-points current amplitude histogram of an excised patch containing 3 IP₃R stimulated with 10 μM IP₃; C and O denote closed and open states. f, Observed and predicted numbers of IP₃R/patch from 109 patches (mean = 1.34) stimulated with 10-100 μM IP₃.

Figure 2. Lone IP₃R are more active than clustered IP₃R at resting cytosolic Ca²⁺

a, Typical records from patches (2 IP₃R/patch) stimulated with IP₃ (μM). b, c, Effect of IP₃ on P_o of patches containing a single IP₃R (b) or on NP_o of patches with 3 IP₃R (c) (n ≥ 4). d, Numbers of IP₃R detected in each patch for each IP₃ concentration (n = 9-25). e, Predicted (ie NP_lone) and observed NP_o for patches containing 1-5 IP₃R (n ≥ 3; n = 2 for 5-IP₃R patch). f, For patches with 3 IP₃R, observed/predicted values are shown for the indicated numbers of simultaneous openings (Supplementary equation 4). g, P_o as a function of the number of IP₃R within a patch after stimulation with 10 μM IP₃ (Supplementary equation 5). h, Effect of IP₃ on P_o for lone IP₃R and IP₃R within multi-IP₃R patches (n ≥ 4).

Figure 3. Reversible clustering of IP₃R by IP₃

a, Numbers of IP₃R detected in patches from naive nuclei (n = 63), after pre-treatment with bath-applied IP₃ (10 μM, ~2 min; n = 88), or the latter after recovery for 8-10 min without IP₃ (n = 40). b-d, Observed and predicted numbers of IP₃R/patch. e, Effects of IP₃ on IP₃R clustering and gating. Clustering is reported by P_o/P_lone for patches with 2 or 3 IP₃R, and gating by NP_o for patches with 2 IP₃R (EC₅₀ = 2.02 ± 0.20 μM). f, τ_o for patches with 2 or 3 IP₃R measured from the duration of single channel openings (blue line, τ_single) or calculated from the duration of openings to the N^th level (red line, τ_calculated = N.τ_o,N). These are compared with τ_o for lone IP₃R (τ_lone). Typical trace is from a patch with 2 IP₃R. g, IP₃ drives IP₃R into small clusters consistent with arrays (grey) formed by IP₃R at high density. Within a cluster, each IP₃R opens independently, but closes more rapidly than a lone IP₃R. h, Typical recording from a patch containing 4 IP₃R with IP₃ released from caged IP₃ in PS by flash photolysis (electrical noise caused by the flash is shown). i, From records similar to h (Supplementary Fig. 8), P_o (from NP_o/N) and τ_o were measured during each 0.5 s interval after the flash (1.5 s for first interval). The ratio (multi-IP₃R patch/lone IP₃R) is shown for both τ_o and P_o. Results (means ± SEM) are from 4 (single) and 7 (multiple, with 2-4 IP₃R/patch) patches.

Figure 4. Clustering retunes Ca²⁺ regulation of IP₃R

a-e, Patches were stimulated with PS containing 10 μM IP₃ and (unless otherwise stated) 1 μM Ca²⁺. a, Typical recording and summary data (n = 5-6) from lone IP₃R show that increasing Ca²⁺ increases P_o by reducing τ_c. b, Observed and expected numbers of IP₃R/patch. c, Observed and predicted NP_o for patches containing 1 or 2 IP₃R and stimulated with 10 μM IP₃ in PS containing 200 nM or 1 μM Ca²⁺ (n = 5-6). d, Typical recording from a patch with 2 IP₃R, enlarged (red) to highlight transitions directly between closed (C) and double open (O2) states. e, Observed and predicted P_o for closed (C) and single (O1) or double openings (O2) for patches with 2 IP₃R (n = 6, Supplementary equations 4, 5). f, Observed and expected durations of events when both IP₃R are simultaneously open (τ_o,2) or closed (τ_c,2) for patches with 2 IP₃R (n = 6, Supplementary equations 6, 7). g, Observed and predicted numbers of transitions to each of the 3 states in a patch with 2 IP₃R (n = 6). h, At resting [Ca²⁺], IP₃ drives IP₃R into small clusters wherein IP₃R gate independently, but with reduced P_o and IP₃ sensitivity. Ca²⁺ reverses the inhibition imposed by clustering, openings within a cluster are more synchronized, and simultaneous openings are prolonged. Clustering primes IP₃R to respond by repressing their activity, and then allowing Ca²⁺ to unleash the coordinated gating of clustered IP₃R (Supplementary Fig. 7).