MOBILE AND IMMOBILE CALCIUM BUFFERS IN BOVINE ADRENAL CHROMAFFIN CELLS

1. The calcium binding capacity (kappa(S)) of bovine chromaffin cells preloaded with fura-2 was measured during nystatin-perforated-patch re cordings. 2. Subsequently, the perforated patch was ruptured to obtain a whole-cell recording situation, and the time course of kappa(S) was monitored during periods of up to one hour. 3. No rapid change (withi n 10-20 s) of kappa(S) was observed upon transition to whole-cell reco rding, as would be expected, if highly mobile organic anions contribut ed significantly to calcium buffering. However, approximately half of the cells investigated displayed a drop in kappa(S) within 2-5 min, in dicative of the loss of soluble Ca2+ binding proteins in the range of 7-20 kDa. 4. The average Ca2+ binding capacity (differential ratio of bound calcium over free calcium) was 9+/-7 (mean+/-S.E.M.) for the poo rly mobile component and 31+/-10 for the fixed component. It was concl uded that a contribution of 7 from highly mobile buffer would have bee n detected, if present. Thus, this value can be considered as an upper bound to highly mobile Ca2+ buffer. 5. Both mobile and fixed calcium binding capacity appeared to have relatively low Ca2+ affinity, since kappa(S) did not change in the range of Ca2+ concentrations between 0. 1 and 3 muM. 6. It was found that cellular autofluorescence and contri butions to fluorescence of non-hydrolysed or compartmentalized dye con tribute a serious error in estimation of kappa(S). 'Balanced loading', a degree of fura-2 loading such that the calcium binding capacity of fura-2 equals cellular calcium binding capacity, minimizes these error s. Also, changes in kappa(S) at the transition from perforated-patch t o whole-cell recording can be most faithfully recorded for similar deg rees of loading in both situations. 7. Nystatin was found unable to ma ke pores from inside of the plasma membrane of chromaffin cells. With careful preparation and storage the diluted nystatin solution maintain ed its high activity of membrane perforation for more than one week. 8 . An equation for the effective diffusion constant for total cytoplasm ic calcium, D(Ca)', was derived, which takes into account fixed buffer and poorly mobile buffer as determined, as well as calcium bound to f ura-2 and some highly mobile buffers. 9. It was concluded that even fo r fura-2 concentrations as small as 50 muM, the dye and possibly some immeasurable contribution of highly mobile buffer dominate the diffusi on process, such that D' varies between approximately 10(-7) cm2 s-1 ( in the absence of fura-2 and of highly mobile buffer) and 4 x 10(-7) c m2 s-1 in the presence of fura-2 and/or highly mobile buffer.