Phasic characteristic of elementary Ca²⁺release sites underlies quantal responses to IP₃

Abstract

Ca 2+ liberation by inositol 1,4,5-trisphosphate (IP 3 ) is quantal', in that low [IP 3 ] causes only partial Ca 2+ release, but further increasing [IP 3 ] evokes more release. This characteristic allows cells to generate graded Ca 2+ signals, but is unexpected, given the regenerative nature of Ca 2+ -induced Ca 2+ release through IP 3 receptors. Two models have been proposed to resolve this paradox: (i) all-or-none Ca 2+ release from heterogeneous stores that empty at varying [IP 3 ]; and (ii) phasic liberation from homogeneously sensitive stores. To discriminate between these hypotheses, we imaged subcellular Ca 2+ puffs evoked by IP 3 in Xenopus oocytes where release sites were functionally uncoupled using EGTA. Puffs were little changed by 300 mM intracellular EGTA, but sites operated autonomously and did not propagate waves. Photoreleased IP 3 generated¯urries of puffsÐ different to the prolonged Ca 2+ elevation following waves in control cellsÐand individual sites responded repeatedly to successive increments of [IP 3 ]. These data support the second hypothesis while refuting the ®rst, and suggest that local Ca 2+ signals exhibit rapid adaptation, different to the slower inhibition following global Ca 2+ waves.

Imaging the quantal substructure of single IP 3 R channel activity during Ca 2+ puffs in intact mammalian cells

“…To then achieve single-channel resolution, we used TIRF imaging of a subset of puff sites located close to the plasma membrane (17), so that rapid binding of Ca 2ϩ to the fast indicator dye within attoliter cytosolic volumes around puff sites yields fluorescence signals that more closely track instantaneous Ca 2ϩ flux through IP 3 R channels (14). Additionally, we gained a further improvement by loading cells with EGTA, a Ca 2ϩ buffer that inhibits wave propagation (17)(18)(19) and accelerates the collapse of the local Ca 2ϩ micro-domain but, because of its slow binding kinetics, minimally perturbs local free [Ca 2ϩ ] within a cluster (14) and has little effect on peak puff amplitudes (17)(18)(19). EGTA is thus…”

Dynamics of excitable elements with time-delayed coupling

“…Opening events can therefore involve single channels, few or all channels in one cluster, or many channels from several clusters. The different release modes are indeed observed experimentally and termed blip, puff (Yao & Parker, 1995;Callamaras & Parker, 2000), or global release, respectively. The fact that the different modes result from different channel synchronization strengths was studied in many publications and can be summarized as follows.…”

“…The latter case can appear due to reduced cooperativity of channels in different clusters. It was found that the addition of an exogenous buffer protein (EGTA) compels the transition from global to local release (Callamaras & Parker, 2000). The transition can be understood from the fact that additional buffer reduces the spatial extent of calcium domains, thereby decreasing the amount of Ca 2+ diffusing to adjacent clusters.…”

“…Instead we here turn to the interesting and not yet understood aspect, that signals decay with different time scales depending on the mode of calcium release (local or global), that is, we study the temporal shape of the release. It was observed that the decay of cluster activity is much faster in the local than in the global mode (Jouaville et al, 1995;Callamaras & Parker, 2000;Dargan & Parker, 2003). If the coupling of clusters in the global mode is indeed stronger, one could then say, that the clusters keep each other active and therefore the activity of the channels decays very slowly.…”

“…The motivation for this method is that in experiments the calcium release is stimulated by increase of IP 3 . IP 3 concentration corresponds to one or more A c c e p t e d m a n u s c r i p t (Callamaras & Parker, 2000) and will be studied in the context of a FHN model in the future.…”