Rapid uptake of calcium, ATP, and inositol 1,4,5‐trisphosphate via cation and anion channels into surface‐derived vesicles from HIT cells containing the inositol 1,4,5‐trisphosphate‐sensitive calcium store

FEBS Letters

Lange, Klaus¹  Brandt, Ursula¹ 

[1] Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-1000 Berlin 33, Germany

关键词: Calcium ion store (HIT cells);    Microvilli;    Cation channel;    Anion channel;    Inositol trisphosphate;    ATP;    DIDS;    4;    4'-diisocyanostilbene-2;    2'-disulfonic acid;    IP3;    inositol 1;    4;    5-trisphosphate;    3-OMG;    3-O-methyl-d-glucose;   

DOI  :  10.1016/0014-5793(93)81074-A

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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].

【 授权许可】

Unknown