Ca(2+) puffs originate from preestablished stable clusters of inositol trisphosphate receptors

Abstract

Intracellular calcium ion (Ca(2+)) signaling crucially depends on the clustered organization of inositol trisphosphate receptors (IP(3)Rs) in the endoplasmic reticulum (ER) membrane. These ligand-gated ion channels liberate Ca(2+) to generate local signals known as Ca(2+) puffs. We tested the hypothesis that IP(3) itself elicits rapid clustering of IP(3)Rs by using flash photolysis of caged IP(3) in conjunction with high-resolution Ca(2+) imaging to monitor the activity and localization of individual IP(3)Rs within intact mammalian cells. Our results indicate that Ca(2+) puffs arising with latencies as short as 100 to 200 ms after photorelease of IP(3) already involve at least four IP(3)R channels, and that this number does not subsequently grow. Moreover, single active IP(3)Rs show limited mobility, and stochastic simulations suggest that aggregation of IP(3)Rs at puff sites by a diffusional trapping mechanism would require many seconds. We thus conclude that puff sites represent preestablished, stable clusters of IP(3)Rs and that functional IP(3)Rs are not readily diffusible within the ER membrane.

Fig. 1

Local Ca²⁺ signals evoked in SH-SY5Y cells following photorelease of i-IP₃. (A) Representative fluorescence traces recorded from 3 puff sites monitored by TIRF microscopy. The records include long baseline sections before a strong (40 ms) photolysis flash (arrow), demonstrating a lack of basal spontaneous activity. Traces show fluorescence ratio changes (ΔF/F₀) of fluo-4, monitored from 3 × 3 pixel (1 × 1 µm) regions of interest. (B) TIRF image shows resting fluorescence of a single fluo-4-loaded SH-SY5Y cell. Circles mark all sites where Ca²⁺ signals were evident following photorelease of i-IP₃. Traces show fluorescence ratio signals (ΔF/F₀) measured from each of the numbered sites marked on the cell image. Asterisks indicate the time of a weak (35 ms) photolysis flash. The inset box shows a single blip at site #14 on enlarged scales.

Fig. 2

Puffs evoked at short latencies following photorelease of i-IP₃ involve similar or greater numbers of IP₃R channels than puffs at longer latencies. (A) Representative fluorescence trace (ΔF/F₀) depicting the first 4 events evoked at a single site following a UV flash. Lower trace shows the first two events (highlighted grey) shown on an expanded time scale showing stepwise transitions in Ca²⁺ fluorescence at approximate multiples of the unitary event level (grey arrows). Solid bars underneath each trace indicate the duration of the UV flashes. (B, C), Plots show peak puff amplitudes as a function of time after onset of the photolysis flash for strong and weak photolysis strengths, respectively. Filled squares denote the amplitude of the first event at each site, with subsequent events at those sites shown as hollow squares. Lines are regression fits to data on semi-logarithmic axes.

Fig. 3

Ca²⁺ signals evoked by photorelease of i-IP₃ in HeLa (A) and rat type I cortical astrocytes (B), recorded with single-channel resolution. The traces at the top show representative events displaying step-wise transitions in Ca²⁺ fluorescence. Signals in both of these cell types were on average smaller than in SH-SY5Y cells, and fewer step levels were evident. Plots show peak amplitudes of first (filled squares) and subsequent (open squares) events as functions of time after a strong (40 ms) photolysis flash. Lines are regressions to data on semi-logarithmic axes.

Fig. 4

Lack of motility of single IP₃Rs. (A) Representative trace showing activity from an apparent ‘lone’ IP₃R following a (200 ms) photolysis flash (asterisk). Fluorescence was measured from a 1 µm square region of interest. (B), The image shows resting fluorescence of a single fluo-4-loaded SH-SY5Y cell, with the locations of all sites showing puffs (multi-channel signals) marked by circles, and with the site from which the trace in A was obtained marked by the box. The inset shows a scatter plot of mean centroid positions (error bars = ± 1SEM) of Ca²⁺ fluorescence during each of the discrete openings (blips) in the trace in A. (C) Bar graph shows the numbers of blips occurring during successive 2s intervals following photorelease of i-IP₃, derived from 15 sites that displayed exclusively single channel activity.

Fig. 5

Modeling cluster formation by a diffusive trap mechanism. Graphs show the numbers of IP₃Rs clustered at each of 7 puff sites in a simulated cell (colored step-wise lines), and the mean number of IP₃Rs per cluster (black curves; average of 50 simulations, 350 puff sites) as functions of time. The simulations model diffusion within a 2-dimensional rectangular cell (10 × 20 µm) in which N IP₃Rs are initially distributed at random and subsequently diffuse with diffusion coefficient D. Seven 'anchoring' sites with diameter L represent puff sites, to which IP₃Rs adhere after colliding. Panels show simulations with the following respective values of D (µm² s⁻¹), N and L (nm) : (A) 0.1, 100, 20; (B) 0,1, 100, 300; (C) 0.1, 1000, 20; (D) 0.03, 100, 20.