In a previous study [K. Lange and U. Brandt (1993) FEBS Lett. 320, 183-188], we showed that the bulk of the ATP-dependent IP3-sensitive Ca2+ store of the hamster insulinoma cell line, HIT-T15, resides in cell surface-derived vesicles most likely of microvillar origin. The origin and orientation of these vesicles suggested that Ca2+ storage is not due to a membrane-located Ca2+ pumping ATPase but rather to ATP-dependent Ca2+-binding within the vesicles. In this case, Ca2+, ATP and IP3 should have free access to the vesicle lumen. This hypothesis was tested. ATP-independent Ca2+ uptake occurred with biphasic kinetics. An initial rapid uptake, which was complete within 30 s, was followed by a slow linear uptake lasting about 10 min. The rapid component was shown by efflux experiments to have an equilibration half-time of about 4 s. This rapid Ca2+ efflux pathway was inhibited by externally applied La3+ (0.1 mM). A similar rapidly equilibrating La3+-sensitive Ca2+ pool was also present in vesicles which had been actively loaded with Ca2+ in the presence of ATP. The intravesicular distribution space of this labile Ca2+ pool was identical with that of the non-metabolizable hexose analogue 3-O-methyl-D-glucose, demonstrating that rapid Ca2+ uptake occurs into a true vesicular water space and is not due to binding. ATP and IP3 were also shown to enter the vesicles by an energy-independent pathway which is inhibited by the anion channel inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM). Both ATP-dependent Ca2+ uptake and IP3-induced Ca2+ release from preloaded vesicles were inhibited by DIDS. These findings clearly demonstrate that (1) the vesicle membrane is permeable to ATP and IP3 via anion channels, and (2) Ca2+ uptake into as well as IP3-induced Ca2+ release from the vesicles occur by passive diffusion through a cation channel which is not regulated by IP3. Consequently, the mechanisms for Ca2+ storage and IP3-induced Ca2+ release must be located in the vesicle lumen. Moreover, the microvillar diffusion-barrier concept, originally proposed for the regulation of hexose transport may also be valid for the receptor-operated regulation of cation and anion influx pathways. Functional coupling of the microvillar Ca2+ stores with the associated cation influx pathway is also strongly supported by the previously demonstrated microvillar shape changes accompanying depletion of the Ca2+ stores by bombesin or thapsigargin in HIT cells [K. Lange and U. Brandt (1992) FEBS Lett. 320, 183-188].
