Tailored protection against plasmalemmal injury by annexins with different Ca2+ sensitivities

The annexins, a family of Ca(2+)- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) triggers plasmalemmal repair. The highly Ca(2+)-sensitive annexin A6 responds faster than annexin A1 to [Ca(2+)](i) elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca(2+) sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca(2+)](i), its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca(2+) sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca(2+)](i). Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca(2+)-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.     

Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells

Upon its genesis during apoptosis, ceramide promotes gross reorganization of the plasma membrane structure involving clustering of signalling molecules and an amplification of vesicle formation, fusion and trafficking. The annexins are a family of proteins, which in the presence of Ca(2+), bind to membranes containing negatively charged phospholipids. Here, we show that ceramide increases affinity of annexin A1-membrane interaction. In the physiologically relevant range of Ca(2+) concentrations, this leads to an increase in the Ca(2+)sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramide-specific antibody, we establish a direct interaction of annexin A1 with areas of the plasma membrane enriched in ceramide (ceramide platforms). In living cells, the intracellular dynamics of annexin A1 match those of plasmalemmal ceramide. Among proteins of the annexin family, the interaction with ceramide platforms is restricted to annexin A1 and is conveyed by its unique N-terminal domain. We demonstrate that intracellular Ca(2+)overload occurring at the conditions of cellular stress induces ceramide production. Using fluorescently tagged annexin A1 as a reporter for ceramide platforms and annexin A6 as a non-selective membrane marker, we visualize ceramide platforms for the first time in living cells and provide evidence for a ceramide-driven segregation and internalization of membrane-associated proteins.     

The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties

The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca(2+)-dependent membrane-binding properties: (1) high Ca(2+) stoichiometry for membrane binding ( approximately 12 mol of Ca(2+)/mol of protein); (2) binding to membranes was very exothermic (> -60 kcal/ mol of protein); and (3) binding to bilayers that were in the liquid-crystal phase but not to bilayers in the gel phase. In contrast, the nontrimer-forming annexin A1 and A2 group had the following Ca(2+)-dependent membrane-binding properties: (1) lower Ca(2+) stoichiometry for membrane binding (相似文献   

Allosterism in membrane binding: a common motif of the annexins?

Annexins are a family of proteins generally described as Ca(2+)-dependent for phospholipid binding. Yet, annexins have a wide variety of binding behaviors and conformational states, some of which are lipid-dependent and Ca(2+)-independent. We present a model that captures the cation and phospholipid binding behavior of the highly conserved core of the annexins. Experimental data for annexins A4 and A5, which have short N-termini, were globally modeled to gain an understanding of how the lipid-binding affinity of the conserved protein core is modulated. Analysis of the binding behavior was achieved through use of the lanthanide Tb(3+) as a Ca(2+) analogue. Binding isotherms were determined experimentally from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb(3+) in the presence or absence of membranes. In the presence of lipid, the affinity of annexin for cation increases, and the binding isotherms change from hyperbolic to weakly sigmoidal. This behavior was modeled by isotherms derived from microscopic binding partition functions. The change from hyperbolic to sigmoidal binding occurs because of an allosteric transition from the annexin solution state to its membrane-associated state. Protein binding to lipid bilayers renders cation binding by annexins cooperative. The two annexin states denote two affinities of the protein for cation, one in the absence and another in the presence of membrane. In the framework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability of the protein to undergo the postulated two-state transition.     

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

Cholesterol enhances phospholipid binding and aggregation of annexins by their core domain

Annexins are Ca(2+)-dependent phospholipid-binding proteins composed of two domains: A conserved core that is responsible for Ca(2+)- and phospholipid-binding, and a variable N-terminal tail. A Ca(2+)-independent annexin 2-membrane association has been shown to be modulated by the presence of cholesterol in the membranes. Herein, the roles of the core and the N-terminal tail on the cholesterol-enhancement of annexin 2 membrane binding and aggregation were studied. The results show that (i) the cholesterol-mediated increase in membrane binding and in the Ca(2+) sensitivity for membrane aggregation were not modified by a N-terminal peptide (residues 15-26), and were conserved in mutants of the N-terminal end (S11 and S25 substitutions); (ii) cholesterol induced an increase in the Ca(2+)-dependent membrane binding and aggregation of the N-terminally truncated protein (Delta 1-29); and (iii) annexins 5 and 6, two proteins with unrelated N-terminal tails and homologous core domains showed a cholesterol-mediated enhancement of the Ca(2+)-dependent binding to membranes. These data indicate that the core domain is responsible for the cholesterol-mediated effects. A model for the cholesterol effect in membrane organisation, annexin binding and aggregation is discussed.     

Calcium-dependent proapoptotic effect of Taenia solium metacestodes annexin B1 on human eosinophils: a novel strategy to prevent host immune response

Annexins are a family of calcium-dependent phospholipid-binding proteins that have been proposed to be involved in a wide range of important biological processes. At present, only a few annexins have been identified in parasites, and the physiological roles of these annexins are obscure. Earlier, we cloned a novel annexin (annexin B1) from Taenia solium metacestodes and found that annexin B1 was detectable in the surrounding host-derived layer with granulomaous infiltration. The objective of this study was to investigate the secretion and physiological function of annexin B1. We expressed a green fluorescent protein-tagged annexin B1 (GFP-anxB1) in living SiHa cells and showed that it was secreted upon stimulation with dexamethasone (Dex). This secretion was not inhibited by brefeldin A but was blocked by pre-treatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Furthermore, we describe for the first time that annexin B1 can bind to the extracellular surface of human eosinophils and produce Ca(2+)-influx. The Ca(2+)-influx induced apoptosis in eosinophils, which was inhibited by pre-loading the Ca(2+) channel blocker 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-metho-xyphenethyl]-1H-imidazole, HCl (SKF-96365). In conclusion, these findings represent direct and substantial evidence for the secretion of annexin B1 by living cells; the apoptosis in eosinophil induced by annexin B1 might be a novel strategy for T. solium metacestodes to prevent the host's immune attack.     

Annexin B12 is a sensor of membrane curvature and undergoes major curvature-dependent structural changes

The regulation of membrane curvature plays an important role in many membrane trafficking and fusion events. Recent studies have begun to identify some of the proteins involved in controlling and sensing the curvature of cellular membranes. A mechanistic understanding of these processes is limited, however, as structural information for the membrane-bound forms of these proteins is scarce. Here, we employed a combination of biochemical and biophysical approaches to study the interaction of annexin B12 with membranes of different curvatures. We observed selective and Ca(2+)-independent binding of annexin B12 to negatively charged vesicles that were either highly curved or that contained lipids with negative intrinsic curvature. This novel curvature-dependent membrane interaction induced major structural rearrangements in the protein and resulted in a backbone fold that was different from that of the well characterized Ca(2+)-dependent membrane-bound form of annexin B12. Following curvature-dependent membrane interaction, the protein retained a predominantly alpha-helical structure but EPR spectroscopy studies of nitroxide side chains placed at selected sites on annexin B12 showed that the protein underwent inside-out refolding that brought previously buried hydrophobic residues into contact with the membrane. These structural changes were reminiscent of those previously observed following Ca(2+)-independent interaction of annexins with membranes at mildly acidic pH, yet they occurred at neutral pH in the presence of curved membranes. The present data demonstrate that annexin B12 is a sensor of membrane curvature and that membrane curvature can trigger large scale conformational changes. We speculate that membrane curvature could be a physiological signal that induces the previously reported Ca(2+)-independent membrane interaction of annexins in vivo.     

Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins

Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.     

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.     

Biophysical and molecular properties of annexin-formed channels

The annexins are water soluble proteins possessing a hydrophilic surface, which belong to a family of proteins which (a) bind ('annex') both calcium and phospholipids, and (b) form voltage-dependent calcium channels within planar lipid bilayers. Annexins types are diverse (94 annexins in 45 species) and they belong to an enormous multigene family that ranges throughout all eukaryotic kingdoms. Although the structure of these proteins is now well known their functional and physiological roles remain largely unknown and circumstantial. Various experimental approaches provided evidence that annexins function as Ca(2+) channels that could act as regulators of membrane fusion. The identity of annexins is derived from the conserved 34 kDa C-terminal domain which comprises four repeats - except for annexin VI, with eight repeats - of a sequence of approximately seventy amino acids, which holds the area known as the 'endonexin fold', with its identifying GXGTDE. Annexins have been placed into three subgroups of (1) tetrad core and short amino terminal, (2) tetrad core and long amino terminal, and (3) octad core and short amino terminal. The repeats are highly conserved, each forming a compact alpha-helical domain comprising five alpha-helices wound in a right-handed superhelix. Four domains are formed, arranged in a nearly flat and cyclical array, with domains I and IV, and II and III respectively forming two tightly organised modules with almost twofold symmetry. A hydrophilic pore lies at the centre of the molecule, forming a prominent ion channel coated with charged and highly conserved residues. The annexin molecule is slightly curved, with both a convex and a concave face. The cation/anion permeability ratios and the selectivity sequence of the ion channels formed by several annexins confirm the selectivity of the annexins for Ca(2+) over other divalent cations, and reveals the importance of structural sites, e.g. amino acid positions 17, 78, 95 and 112 for the identification of the ion channel's position, function and regulation. Some are sensitive to low doses of the phenothiazine drugs, trifluoperazine (an anti-schizophrenia drug) and promethazine (anti nausea drug) La(3+) and Cd(2+), (blockers of voltage-gated Ca(2+) channels) nifedipine (an inhibitor of non-activating Ca(2+) channels). There are two main competing models used to explain in vitro ion channel activity of annexins: one involves changes in the conductance of ion via electrostatic disturbance of the membrane surface; the other involves a much more extensive alteration in protein structure and a correspondingly deeper penetration into the membrane.     

Calcium signaling and annexins

The annexins, are a family of calcium ion (Ca2+)-binding proteins whose physiological functions are poorly understood. Although many diverse functions have been proposed for these proteins, such as in vesicle trafficking, this review focuses on their proposed roles as Ca2+ or other ion channels, or as intracellular ion channel regulators. Such ideas are founded mainly on in vitro and structural analyses, but there is increasing evidence that at least some members of this protein family may indeed play a part in intracellular Ca2+ signaling by acting both as atypical ion channels and as modulators of ion channel activity. This review first introduces the annexin family, then discusses intracellular localization, developmental regulation, and modes of membrane association of annexins, which suggest roles in Ca2+ homeostasis. Finally, it examines the structural and electrophysiological data that argue for key roles for annexins in the control of ion fluxes.     

Annexins V and XII insert into bilayers at mildly acidic pH and form ion channels

The functional hallmark of annexins is the ability to bind to the surface of phospholipid membranes in a reversible, Ca(2+)-dependent manner. We now report that human annexin V and hydra annexin XII reversibly bound to phospholipid vesicles in the absence of Ca(2+) at low pH; half-maximal vesicle association occurred at pH 5.3 and 5. 8, respectively. The following biochemical data support the hypothesis that these annexins insert into bilayers at mildly acidic pH. First, a photoactivatable reagent (3-trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine) which selectively labels proteins exposed to the hydrophobic domain of bilayers reacted with these annexins at pH 5.0 and below but not at neutral pH. Second, in a Triton X-114 partitioning assay, annexins V and XII act as integral membrane proteins at low pH and as hydrophilic proteins at neutral pH; in the presence of phospholipids half-maximal partitioning into detergent occurred at pH approximately 5.0. Finally, annexin V or XII formed single channels in phospholipid bilayers at low pH but not at neutral pH. A model is discussed in which the concentrations of H(+) and Ca(2+) regulate the reversible conversion of three forms of annexins-soluble, peripheral membrane, and transmembrane.     

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca(2+)-dependent and Ca(2+)-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.     

Annexins: linking Ca2+ signalling to membrane dynamics

Eukaryotic cells contain various Ca(2+)-effector proteins that mediate cellular responses to changes in intracellular Ca(2+) levels. A unique class of these proteins - annexins - can bind to certain membrane phospholipids in a Ca(2+)-dependent manner, providing a link between Ca(2+) signalling and membrane functions. By forming networks on the membrane surface, annexins can function as organizers of membrane domains and membrane-recruitment platforms for proteins with which they interact. These and related properties enable annexins to participate in several otherwise unrelated events that range from membrane dynamics to cell differentiation and migration.     

Ca(2+)-dependent annexin self-association on membrane surfaces

Annexin self-association was studied with 90 degrees light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca(2+)-dependent manner in solution. However, this activity was quite labile and, especially for p32 and p67, was not consistently observed. When bound to chromaffin granule membranes, the three proteins consistently self-associated and did so at Ca2+ levels (pCa 5.0-4.5) approximately 10-fold lower than required when in solution. Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine (1:1 or 1:3) were less effective at supporting annexin polymerization than were those containing phosphatidylserine and phosphatidylcholine (1:0, 1:1, or 1:3). The annexins bound chromaffin granule membranes in a positively cooperative manner under conditions where annexin self-association was observed, and both phenomena were inhibited by trifluoperazine. Ca(2+)-dependent chromaffin granule membrane aggregation, induced by p32 or synexin, was associated with intermembrane annexin polymerization at Ca2+ levels less than pCa 4, but not at higher Ca2+ concentrations, suggesting that annexin self-association may be necessary for membrane contact at low Ca2+ levels but not at higher Ca2+ levels where the protein may bind two membranes as a monomer.     

Ca(2+)-dependent association of S100A6 (Calcyclin) with the plasma membrane and the nuclear envelope.

Expression of S100A6 (Calcyclin), a member of the S100 family and of Zn(2+)-binding proteins is elevated in a number of malignant tumors. In vitro the protein associates with several actin-binding proteins and annexins in a Ca(2+)-dependent manner. We have now studied the subcellular localization of S100A6 using a new, specific monoclonal antibody. Immunofluorescence microscopy of unfixed, ultrathin, frozen sections demonstrated a dual localization of S100A6 at the nuclear envelope and the plasma membrane of porcine smooth muscle only in the presence of Ca(2+). The same localization was found by immunofluorescence and immunogold electron microscopy as well as by confocal laser scanning microscopy with cultured, fixed, human CaKi-2 and porcine ST interphase cells. Upon cell division, however, S100A6 was found exclusively in the cytoplasm. Cell fractionation studies showed that S100A6 was present in the microsomal fraction in the presence of Ca(2+) and was released from this fraction by the addition of EGTA/EDTA but not by Triton X-100. The data demonstrate that S100A6 is localized both at the plasma membrane and the nuclear envelope in vivo and suggest a Ca(2+)-dependent interaction with annexins or other components of the nuclear envelope.     

Annexin V counteracts apoptosis while inducing Ca(2+) influx in human lymphocytic T cells

We have previously shown that when annexin V is present during the execution of a cell death program, apoptosis is delayed. This is reflected by the inhibition of DNA cleavage and of the release of apoptotic membrane particles, and by reduction of the proteolytic processing of caspase-3. Here, we have studied the mechanism(s) through which annexin V counteracts apoptosis in the human CEM T cell line. The degree of apoptosis inhibition was associated with an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). Reduction of the extracellular Ca(2+) concentration by EGTA abolished the anti-apoptotic effect, suggesting that annexin V favors Ca(2+) influx and that Ca(2+) acts as an inhibitor rather than an activator of apoptosis in CEM T cells. The effects on apoptosis and [Ca(2+)](i) of several modified annexins with different electrophysiological properties indicate that the N-terminal domain of annexin V is necessary for the Ca(2+)-dependent anti-apoptotic action of annexin V. These results suggest that annexin V regulates membrane Ca(2+) permeability and is protective against apoptosis by increasing [Ca(2+)](i) in CEM T cells.     

Annexin VI participates in the formation of a reversible, membrane-cytoskeleton complex in smooth muscle cells

The plasmalemma of smooth muscle cells is periodically banded. This arrangement ensures efficient transmission of contractile activity, via the firm, actin-anchoring regions, while the more elastic caveolae-containing "hinge" regions facilitate rapid cellular adaptation to changes in cell length. Since cellular mechanics are undoubtedly regulated by components of the membrane and cytoskeleton, we have investigated the potential role played by annexins (a family of phospholipid- and actin-binding, Ca(2+)-regulated proteins) in regulating sarcolemmal organization. Stimulation of smooth muscle cells elicited a relocation of annexin VI from the cytoplasm to the plasmalemma. In smooth, but not in striated muscle extracts, annexins II and VI coprecipitated with actomyosin and the caveolar fraction of the sarcolemma at elevated Ca(2+) concentrations. Recombination of actomyosin, annexins, and caveolar lipids in the presence of Ca(2+) led to formation of a structured precipitate. Participation of all 3 components was required, indicating that a Ca(2+)-dependent, cytoskeleton-membrane complex had been generated. This association, which occurred at physiological Ca(2+) concentrations, corroborates our biochemical fractionation and immunohistochemical findings and suggests that annexins play a role in regulating sarcolemmal organization during smooth muscle contraction.     

Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2

S100 proteins are a group of EF-hand calcium-signaling proteins, many of which interact with members of the calcium- and phospholipid-binding annexin family of proteins. This calcium-sensitive interaction enables two neighboring membrane surfaces, complexed to different annexin proteins, to be brought into close proximity for membrane reorganization, using the S100 protein as a bridging molecule. S100A11 and S100A10 are two members of the S100 family found to interact with the N-termini of annexins A1 and A2, respectively. Despite the high degree of structural similarity between these two complexes and the sequences of the peptides, earlier studies have shown that there is little or no cross-reactivity between these two S100s and the annexin peptides. In the current work the specificity and the affinity of the interaction of the N-terminal sequences of annexins A1 and A2 with Ca2+-S100A11 were investigated. Through the use of alanine-scanning peptide array experiments and NMR spectroscopy, an approximate 5-fold tighter interaction was identified between Ca2+-S100A11 and annexin A2 (approximately 3 microM) compared to annexin A1 (approximately 15 microM). Chemical shift mapping revealed that the binding site for annexin A2 on S100A11 was similar to that observed for the annexin A1 but with distinct differences involving the C-terminus of the annexin A2 peptide. In addition, kinetic measurements based on NMR titration data showed that annexin A2 binding to Ca2+-S100A11 occurs at a comparable rate (approximately 120 s(-1)) to that observed for membrane fusion processes such as endo- and exocytosis.