The annexins are a family of Ca
2+- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca
2+]
i or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca
2+ entry (SOCE), but did not influence the rates of Ca
2+ extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca
2+ entry as long as [Ca
2+]
i was below the threshold of annexin A6-membrane translocation. However, when [Ca
2+]
i reached the levels necessary for the Ca
2+-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca
2+ entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca
2+ entry, with consequences for the rates of cell proliferation.Calcium entry into cells either through voltage- or receptor-operated channels, or following the depletion of intracellular stores is a major factor in maintaining intracellular Ca
2+ homeostasis. Resting [Ca
2+]
i is low (∼100 n
m compared with extracellular [Ca
2+]
ex of 1.2 m
m) and can be rapidly increased by inositol triphosphate-mediated release from the intracellular Ca
2+ stores (mostly endoplasmic reticulum (ER)
3), or by channel-mediated influx across the plasma membrane (PM). Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca
2+ entry in non-excitable cells (
1), and the recent discovery of Orai1 and STIM provided the missing link between the Ca
2+-release activated current (I
CRAC) and the ER Ca
2+ sensor (
2–
4). Translocation of STIM within the ER, accumulation in punctae at the sites of contact with PM and activation of Ca
2+ channels have been proposed as a model of its regulation of Orai1 activity (
5,
6). However, many details of the functional STIM-Orai1 protein complex and its regulation remain to be elucidated. The actin cytoskeleton plays a major role in the regulation of SOCE, possibly by influencing the function of ion channels or by interfering with the interaction between STIM and Orai1 (
7–
9). However, the proteins connecting the actin cytoskeleton and SOCE activity at the PM have yet to be identified.The annexins are a multigene family of Ca
2+- and phospholipid-binding proteins, which have been implicated in many Ca
2+-regulated processes. Their C-terminal core is evolutionarily conserved and contains Ca
2+-binding sites, their N-terminal tails are unique and enable the protein to interact with distinct cytoplasmic partners. At low [Ca
2+]
i, annexins are diffusely distributed throughout the cytosol, however, after stimulation resulting in the increase of [Ca
2+]
i, annexins are targeted to distinct subcellular membrane locations, such as the PM, endosomes, or secretory vesicles (
10). Annexins are involved in the processes of vesicle trafficking, cell division, apoptosis, calcium signaling, and growth regulation (
11), and frequent changes in expression levels of annexins are observed in disease (
12,
13). Previously, using biochemical methods and imaging of fluorescent protein-tagged annexins in live cells, we demonstrated that annexins A1, A2, A4, and A6 interacted with the PM as well as with internal membrane systems in a highly coordinated manner (
10,
14). In addition, there is evidence of Ca
2+-independent membrane association of several annexins, including annexin A6 (
15–
19); some of which point to the existence of pH-dependent binding mechanisms (
20–
22). Given the fact that several annexins are present within any one cell, it is likely that they form a [Ca
2+] and pH sensing system, with a regulatory influence on other signaling pathways.The role of annexins as regulators of ion channel activity has been addressed previously (
23–
25). In particular, annexin A6 has been implicated in regulation of the sarcoplasmic reticulum ryanodine-sensitive Ca
2+ channel (
25), the neuronal K
+ and Ca
2+ channels (
26), and the cardiac Na
+/Ca
2+ exchanger (
27). Cardiac-specific overexpression of annexin A6 resulted in lower basal [Ca
2+], a depression of [Ca
2+]
i transients and impaired cardiomyocyte contractility (
28). In contrast, the cardiomyocytes from the annexin A6 null-mutant mice showed increased contractility and accelerated Ca
2+ clearance (
29). Consistent with its role in mediating the intracellular Ca
2+ signals, especially Ca
2+ influx, ectopic overexpression of annexin A6 in A431 cells, which lack endogenous annexin A6, resulted in inhibition of EGF-dependent Ca
2+ entry (
30).The difficulty of investigating the influence of annexins on signaling events occurring at the PM lies in the transient and reversible nature of their Ca
2+ and pH-dependent lipid binding. Although the intracellular Ca
2+ increase following receptor activation or Ca
2+ influx promotes the association of the Ca
2+-sensitive annexins A2 and A6 with the PM, the proteins quickly resume their cytoplasmic localization upon restoration of the basal [Ca
2+]
i (
14). Therefore, to investigate the effects of membrane-associated annexins on Ca
2+ homeostasis and the cell signaling machinery, we aimed to develop a model system allowing for a constitutive membrane association of annexins. Here we used the PM-anchoring sequences of the H- and K-Ras proteins to target annexins A6 and A1 to the PM independently of [Ca
2+]. The Ras GTPases are resident at the inner leaflet of the PM and function as molecular switches (
31). The C-terminal 9 amino acids of H- and N-Ras and the C-terminal 14 amino acids of K-Ras comprise the signal sequences for membrane anchoring of Ras isoforms (
32). Although the palmitoylation and farnesylation of the C terminus of H-Ras (tH) serves as a targeting signal for predominantly cholesterol-rich membrane microdomains at the PM (lipid rafts/caveolae) (
33), the polybasic group and the lipid anchor of K-Ras (tK) ensures the association of K-Ras with cholesterol-poor PM membrane domains. Importantly, these minimal C-terminal amino acid sequences are sufficient to target heterologous proteins, for example GFP, to different microdomains at the PM and influence their trafficking (
34).In the present study we fused annexins A6, A2, and A1 with fluorescent proteins and introduced the PM-anchoring sequences of either H-Ras (annexin-tH) or K-Ras (annexin-tK) at the C termini of the fusion constructs. We demonstrate that the constitutive PM localization of annexin A6 results in down-regulation of store-operated Ca
2+ entry. Expression of membrane-anchored annexin A6 causes an accumulation of the cortical F-actin, and cytoskeletal destabilization with latrunculin A abolishes the inhibitory effect of PM-anchored annexin A6 on SOCE. Taken together, our results implicate annexin A6 in the maintenance of intracellular Ca
2+ homeostasis via actin-dependent regulation of Ca
2+ entry.
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