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Calcium storage and release properties of F‐actin: evidence for the involvement of F‐actin in cellular calcium signaling
Author(s) -
Lange Klaus,
Brandt Ursula
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)01025-3
Subject(s) - divalent , actin , phalloidin , biophysics , chemistry , calcium , biochemistry , cytoskeleton , biology , cell , organic chemistry
Preceding studies have shown that the bulk of the ATP‐dependent, inositol 1,4,5‐trisphosphate (IP 3 )‐sensitive Ca 2+ 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 Ca 2+ , ATP, and IP 3 occur through cation and anion channels, respectively, suggesting that Ca 2+ storage is due to ATP‐dependent Ca 2+ binding to an intravesicular component. Here we demonstrate that F‐actin may be a possible candidate for this function. ATP‐actin monomers bind Ca 2+ 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 Ca 2+ can be released by depolymerization and dissociation from Ca 2+ ‐ADP‐actin monomers ( K d = 375 nM). We now provide additional evidence for the possible involvement of actin in Ca 2+ storage. (1) Preincubation of surface‐derived Ca 2+ ‐storing vesicles from HIT cells with the F‐actin stabilizer, phalloidin, strongly inhibited ATP‐dependent Ca 2+ uptake, reducing the IP 3 ‐sensitive Ca 2+ pool by 70%. Phalloidin, when added after the loading process, affected neither the amount of stored Ca 2+ nor IP 3 action on the store. (2) F‐actin polymerized in the presence of Mg 2+ in nominally Ca 2+ ‐free buffer still contained about half of the high affinity sites occupied with Ca 2+ (Mg/Ca‐F‐actin). (3) Using the fura‐2 technique, we found that in the presence of ATP, Mg/Ca‐F‐actin incorporated free Ca 2+ at a relatively low rate. Short pulses of ultrasound (3–10s) strongly accelerated Ca 2+ uptake, decreasing free Ca 2+ from 500 nM to below 100 nM. (4) In the presence of physiological levels of Mg 2+ (0.5 mM), sonication liberated large amounts of Ca 2+ from Mg/Ca‐F‐actin. (5) Ca‐F‐actin released bound Ca 2+ at a very slow rate. Short ultrasonic pulses rapidly elevated free Ca 2+ from about 50 nM up to 500 nM. (6) Small amounts of profilin, an actin‐binding protein, released Ca 2+ both from Ca‐and Mg/Ca‐F‐actin and also inhibited uptake of Ca 2+ into Mg/Ca‐F‐actin. (7) Phalloidin completely inhibited Ca‐uptake into Mg/Ca‐F‐actin even during ultrasonic treatment. These findings suggest that Ca 2+ 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 Ca 2+ 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 Ca 2+ signaling system for living cells remains to be established.