A PHOGRIN-AEQUORIN CHIMERA TO IMAGE FREE CA2+ IN THE VICINITY OF SECRETORY GRANULES

Microdomains of high Ca2+ concentration ([Ca2+]) may be critical to th e control of intracellular processes such as secretion and metabolism without compromising other cell functions. To explore changes in [Ca2] in the outer mantle (< 30 nm deep) that surrounds the surface of den se-core secretory granules, we have designed a recombinant chimaera be tween the granule protein phogrin and aequorin. When expressed in popu lations of insulin-secreting MIN6 or phaeochromocytoma PC12 cells, the chimaera was targeted to secretory granules as expected. The recombin ant protein reported a similar [Ca2+] at the granule surface to that i n the bulk cytosol, measured with untargeted aequorin. This was the ca se both at rest ([Ca2+] = 80-120 nM) and after stimulation with agents that provoke Ca2+ entry or Ca2+ mobilization from intracellular pools , and during activated secretion. Thus depolarization of MIN6 cell pop ulations with high K+ increased [Ca2+] both in the bulk cytosol and cl ose to the granules to approx. 4 mu M, with near-identical kinetics of increase and recovery. Similarly, stimulation of PC12 cells with ATP provoked an increase in [Ca2+] in either domain to 1.3 mu M. These dat a argue that, in MIN6 and PC12 neuroendocrine cells (i) significant mo bilization of Ca2+ from most secretory granules probably does not occu r during activated Ca2+ influx or mobilization of internal Ca2+ stores , and (ii) agonist-stimulated Ca2+-dependent secretion can occur witho ut development of a large gradient of [Ca2+] between the surface of mo st secretory vesicles and the rest of the cytosol.