Calcium storage and release properties of F‐actin: evidence for the involvement of F‐actin in cellular calcium signaling

FEBS Letters

Lange, Klaus¹  Brandt, Ursula¹ 

[1] Institut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany

关键词: Calcium ion store;    HIT cell;    Microvilli;    F-actin;    Profilin;    Phalloidin;   

DOI  :  10.1016/0014-5793(96)01025-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Preceding studies have shown that the bulk of the ATP-dependent, inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ store of hamster insulinoma (HIT) cells is located in microvilli on the cell surface. Similar results were obtained with isolated rat hepatocytes. Moreover, in vesicles of microvillar origin, passive fluxes of Ca²⁺, ATP, and IP₃ occur through cation and anion channels, respectively, suggesting that Ca²⁺ storage is due to ATP-dependent Ca²⁺ binding to an intravesicular component. Here we demonstrate that F-actin may be a possible candidate for this function. ATP-actin monomers bind Ca²⁺ with high affinity (K _d = 2−8 nM) to their divalent cation binding sites. Polymerization of actin monomers decreases the rate constant for divalent cation exchange at this binding site by more than 3 orders of magnitude rendering bound cations nearly unavailable. F-actin-bound Ca²⁺ can be released by depolymerization and dissociation from Ca²⁺-ADP-actin monomers (K _d = 375 nM). We now provide additional evidence for the possible involvement of actin in Ca²⁺ storage. (1) Preincubation of surface-derived Ca²⁺-storing vesicles from HIT cells with the F-actin stabilizer, phalloidin, strongly inhibited ATP-dependent Ca²⁺ uptake, reducing the IP₃-sensitive Ca²⁺ pool by 70%. Phalloidin, when added after the loading process, affected neither the amount of stored Ca²⁺ nor IP₃ action on the store. (2) F-actin polymerized in the presence of Mg²⁺ in nominally Ca²⁺-free buffer still contained about half of the high affinity sites occupied with Ca²⁺ (Mg/Ca-F-actin). (3) Using the fura-2 technique, we found that in the presence of ATP, Mg/Ca-F-actin incorporated free Ca²⁺ at a relatively low rate. Short pulses of ultrasound (3–10s) strongly accelerated Ca²⁺ uptake, decreasing free Ca²⁺ from 500 nM to below 100 nM. (4) In the presence of physiological levels of Mg²⁺ (0.5 mM), sonication liberated large amounts of Ca²⁺ from Mg/Ca-F-actin. (5) Ca-F-actin released bound Ca²⁺ at a very slow rate. Short ultrasonic pulses rapidly elevated free Ca²⁺ from about 50 nM up to 500 nM. (6) Small amounts of profilin, an actin-binding protein, released Ca²⁺ both from Ca-and Mg/Ca-F-actin and also inhibited uptake of Ca²⁺ into Mg/Ca-F-actin. (7) Phalloidin completely inhibited Ca-uptake into Mg/Ca-F-actin even during ultrasonic treatment. These findings suggest that Ca²⁺ storage may occur by addition of Ca-ATP-actin monomers to reactive ends of the polymer and emptying of this store by profilin-stimulated release of Ca-ADP-actin. Thus, receptor-operated Ca²⁺ signaling, initiated by phospholipase C activation, may proceed via the well-known phosphatidylinositol phosphate-regulated profilin/gelsolin pathway of actin reorganization/depolemerization. The importance of the proposed microvillar Ca²⁺ signaling system for living cells remains to be established.

【 授权许可】

Unknown