We describe a finite-element model of mast cell calcium dynamics that incorporates the endoplasmic reticulum's complex geometry. The model is built upon a three-dimensional reconstruction of the endoplasmic reticulum (ER) from an electron tomographic tilt series. Tetrahedral meshes provide volumetric representations of the ER lumen, ER membrane, cytoplasm, and plasma membrane. The reaction-diffusion model simultaneously tracks changes in cytoplasmic and ER intraluminal calcium concentrations and includes luminal and cytoplasmic protein buffers. Transport fluxes via PMCA, SERCA, ER leakage, and Type II IP^sub 3^ receptors are also represented. Unique features of the model include stochastic behavior of IP^sub 3^ receptor calcium channels and comparisons of channel open times when diffusely distributed or aggregated in clusters on the ER surface. Simulations show that IP^sub 3^R channels in close proximity modulate activity of their neighbors through local Ca^sup 2+^ feedback effects. Cytoplasmic calcium levels rise higher, and ER luminal calcium concentrations drop lower, after IP^sub 3^-mediated release from receptors in the diffuse configuration. Simulation results also suggest that the buffering capacity of the ER, and not restricted diffusion, is the predominant factor influencing average luminal calcium concentrations.

The mobilization of calcium is a vital step in mast cell activation. Cross linking of high affinity IgE receptors initiates a tyrosine kinase cascade that activates phospholipase Cγ isoforms and leads to elevated levels of inositol 1.4.5-trisphosphate (IP^sub 3^) (I). Phosphatidylinositol 3-kinase lipid products enhance PLCy activity and are required for maximal IP^sub 3^ synthesis (1-4). Under optimal cross-linking conditions, intracellular Ca^sup 2+^ stores are rapidly depleted and do not refill tor minutes (5). Concomitant Ca^sup 2+^ influx supports a persistent elevation in cytoplasmic Ca^sup 2+^. The sustained phase of Ca^sup 2+^ influx occurs primarily via the capadtutive Ca^sup 2+^ pathway (6) and, paradoxically, is the phase of the response that is most dramatically affected by PI 3-kinase inhibition (1.4). Sustained elevations in cytoplasmic Ca^sup 2+^ are absolutely required for secretion of histamine, serotonin, and other preformed mediators of the allergic response (7).

Intracellular calcium stores in nonmuscle cells are principally released by IP^sub 3^ receptors, of which there are three closely related types (8). In 1998 we reported that Type II IP^sub 3^ receptors of RBL-2H3 cells, a mast tumor cell line, redistribute into large clusters in the endoplasmic reticulum (ER) after short periods of elevated intracellular calcium (9). Similar results were observed for Type 1 receptors in rat pancreatoma cells and for Type II receptors in hamster lung fibroblasts, suggesting that the induction of IP^sub 3^ receptor clustering is a common feature of this family of channels. We hypothesized that redistribution of receptors modulates Ca^sup 2+^ release from the ER. To address this issue, we have created a three-dimensional modeling environment that incorporates a realistic ER geometry and tracks free and hound calcium based upon reaction-diffusion rates and spatial constraints.

The modeling of an entire cell has been proposed as a grand challenge for this century (10). Modeling platforms such as Virtual Cell (11) and Mcell (12,13) represent important advances in this direction. Here we describe the development of a model cell that houses a geometric reconstruction of the ER and is populated with basic components of the calcium response pathway in most nonexcitable cells, including IP^sub 3^ receptors, SERCA and PMCA ATP-driven pumps, and calcium buffering proteins in the cytosol and ER lumen. A simpler ER disk model was also developed for comparison to the complex ER geometry model and for less costly pilot simulations. In both models, individual domains representing membranes, cytosol, and ER lumen are composed of tetrahedrons, providing a framework for solving multispecies diffusion/reaction equations at millisecond timescales. We report computational support for the concept that clustering influences channel behavior. Mobilization of clustered IP^sub 3^ receptors can result in modest gradients of calcium within the ER lumen during the initial phase of stores release though IP^sub 3^ receptors. However, its most important effect is to dramatically reduce the open channel probability and the release of stored calcium. Luminal buffers effectively minimize gradients, despite the potential for restricted diffusion in tight spaces within the ER. Hence, the principal consequence of stimulus-coupled receptor clustering may he to reduce IP^sub 3^R channel activity, protecting luminal calcium levels when cells are repeatedly stimulated.