Quantifying calcium fluxes underlying calcium puffs in Xenopus laevis oocytes

Abstract

SummaryWe determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al., 2005 [1]. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca 2+ release comes from a region of width ~ 450 nm, that the release duration is peaked around 18ms and that the underlying Ca 2+ currents range between 0.12 and 0.95pA. All these parameters are independent of IP 3 concentration. We explore what distributions of channels that open during a puff, N p , and what relations between current and number of open channels, I(N p ), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al, 2006 [2]. To this end, we use simple "mean field" models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I(N p ) ~N p for small N p and I(N p )~N p 1/α (α>1) for large N p , provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of ~400 puffs obtained using the dye Oregon green.

Mode switching of Inositol 1,4,5-trisphosphate receptor channel shapes the Spatiotemporal scales of Ca2+ signals

“…I , Z = 2 and F represent the Ca 2+ current passing through the channel, valence of Ca 2+ and Faraday's constant respectively. Previous studies have concluded that a single InsP 3 R channel releases a current of about 0.05-0.5 pA [8,19,20]. Throughout this manuscript we will assume a standard single-channel current of I = 0.05 pA when the channel is open, and zero otherwise.…”

“…Experimental observations suggest a simultaneous opening of 6-10 channels during a Ca 2+ puff (a Ca 2+ release event caused by a simultaneous opening of more than one channel in a cluster) [5,6] and a typical cluster diameter of about 0.5 μm [5,7,8]. We assume that the maximum number of simultaneously open channels ever observed at a given puff site represents the number of channels within the cluster giving a mean inter-channel spacing of about 100-150 nm depending on the size and number of channels within a cluster.…”

Observable effects of Ca 2+ buffers on local Ca 2+ signals

“…Based on previous analyses (Bruno et al 2010), the model assumes that a cluster occupies a circle of fixed radius, R = 250 nm, and that N IP 3 Rs are randomly distributed inside it with uniform probability. The model has been developed for data collected from similar-type clusters, so that the value of N is also fixed.…”

“…The aim is to determine how r inf should be varied in the model to account for the presence of these added buffers. To this end, we simulate the reaction-diffusion system that models the dynamics of cytosolic Ca 2+ as described in Solovey et al (2008) in the presence of one open IP 3 R. An open IP 3 R is considered to be a point source that releases a constant Ca 2+ current that was estimated from experimental data to be 0.1 pA (Bruno et al 2010). The reaction-diffusion system (Solovey et al 2008) also includes a cytosolic Ca 2+ indicator dye and an exogenous buffer, either ethylene glycol tetraacetic acid (EGTA) or bis(2-aminophenoxy)ethane tetraacetic acid (BAPTA).…”

“…The combination of this inhomogeneity and of CICR gives rise to a large variety of intracellular Ca 2+ signals, which go from local ones such as 'blips' (Ca 2+ release through a single IP 3 R) and 'puffs' (Ca 2+ release through several IP 3 Rs in a cluster) to waves that propagate throughout the cell . These signals have been observed using fluorescence microscopy and Ca 2+ -sensitive dyes (Bootman et al 1997;Callamaras et al 1998;Sun et al 1998), and various models have been presented to interpret the observations (Swillens et al 1999;Shuai et al 2006;Swaminathan et al 2009;Thul et al 2009;Bruno et al 2010). The use of total internal reflection fluorescence (TIRF) microscopy and a fast charge-coupled device camera in intact mammalian cells represents an experimental breakthrough that is giving more direct information on intracluster properties (Smith & Parker 2009).…”