2-DIMENSIONAL DETERMINATION OF THE CELLULAR CA2+ BINDING IN BOVINE CHROMAFFIN CELLS

The spatiotemporal profile of intracellular calcium signals is determi ned by the flux of calcium ions across different biological membranes as well as by the diffusional mobility of calcium and different calciu m buffers in the cell. To arrive at a quantitative understanding of th e determinants of these signals, one needs to dissociate the flux cont ribution from the redistribution and buffering of calcium. Since the c ytosol can be heterogeneous with respect to its calcium buffering prop erty, it is essential to assess this property in a spatially resolved manner. In this paper we report on two different methods to estimate t he cellular calcium binding of bovine adrenal chromaffin cells. In the first method, we use voltage-dependent calcium channels as a source t o generate calcium gradients in the cytosol. Using imaging techniques, we monitor the dissipation of these gradients to estimate local appar ent calcium diffusion coefficients and, from these, local calcium bind ing ratios. This approach requires a very high signal-to-noise ratio o f the calcium measurement and can be used when well-defined calcium gr adients can be generated throughout the cell. In the second method, we overcome these problems by using calcium-loaded DM-nitrophen as a lig ht-dependent calcium source to homogeneously and quantitatively releas e calcium in the cytosol, By measuring [Ca2+] directly before and afte r the photorelease process and knowing the total amount of calcium bei ng released photolytically, we get an estimate of the fraction of calc ium ions which does not appear as free calcium and hence must be bound to either the indicator dye or the endogenous calcium buffer. This fi nally results in a two-dimensional map of the distribution of the immo bile endogenous calcium buffer. We did not observe significant variati ons of the cellular calcium binding at a spatial resolution of similar to 2 mu m. Furthermore, the calcium binding is not reduced by increas ing the resting [Ca2+] to levels as high as 1.1 mu M. This is indicati ve of a low calcium affinity of the corresponding buffers and is in ag reement with a recent report on the affinity of these buffers (Xu, T., M. Naraghi, H. Kang, and E. Neher. 1997. Biophys. J. 73:532-545). In contrast to the homogeneous distribution of the calcium buffers, the a pparent calcium diffusion coefficient did show inhomogeneities, which can be attributed to restricted diffusion at the nuclear envelope and to rim effects at the cell membrane.