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Acidic pH-induced folding of annexin VI is a prerequisite for its insertion into lipid bilayers and formation of ion channels by the protein molecules¹

MARCIN GOLCZAK^*, ANNA KICINSKA^*, JOANNA BANDOROWICZ-PIKULA^*, RENE BUCHET, ADAM SZEWCZYK^* and SLAWOMIR PIKULA^*2

Departments of
^* Cellular Biochemistry, Nencki Institute of Experimental Biology, 02–093 Warsaw, Poland; and
Laboratoire Physico-Chimie Biologique, Université Claude Bernard, Lyon I, UFR de Chimie-Biochimie, CNRS UMR 5013, Villeurbanne, France

²Correspondence: Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur St., 02–093 Warsaw, Poland. E-mail: slawek{at}nencki.gov.pl

SPECIFIC AIMS

We tested the hypothesis that annexin VI (AnxVI), a member of a family of Ca²⁺ and lipid binding proteins, can interact with membranes in a Ca²⁺-independent manner and behave as a membrane integral protein. For this purpose, we investigated the consequences of AnxVI pH-dependent binding to lipid membranes, such as the formation of ion channels by the annexin molecules and pH-induced conformational changes of AnxVI, to understand some functional aspects of the protein in vivo, particularly under pathological conditions, connected with local fluctuations of pH.

PRINCIPAL FINDINGS

1. The binding of AnxVI to phospholipids in a Ca²⁺-insensitive, pH-dependent manner constitutes an alternative to the Ca²⁺ bridging mechanism of interaction of annexins with membranes
The Ca²⁺-independent binding of AnxVI to asolectin liposomes is stimulated by acidic pH; half-maximal binding occurred at pH 5.3. The lipid composition of liposomes is not crucial for AnxVI–lipid interactions at low pH. The binding at lower pH is inhibited by increasing the pH above 7.0 in the absence of Ca²⁺. A reciprocal experiment, i.e., AnxVI bound to liposomes at pH 3.0 and then the pH of assay medium raised to 7.4, revealed that AnxVI did not dissociate from the membrane even after 60 min incubation at room temperature. The results obtained with liposomes were confirmed with the use of a monolayer technique. At pH 4.6 and in the absence of Ca²⁺, AnxVI increased the of asolectin monolayers at moderate initial (17–19 mN/m), whereas at a pH above 6.8 no such effect was observed. An increased of asolectin monolayers occurred at nanomolar AnxVI concentrations (K_1/2 15 nM AnxVI). This can be interpreted as a substantial penetration of AnxVI molecules into asolectin monolayers at acidic pH.

2. At mildly acidic pH, AnxVI forms voltage-dependent ion channels in lipid planar bilayers, revealing no specificity for the transported ions
To monitor Ca²⁺-independent pH-sensitive interactions of AnxVI with membranes, the ability of AnxVI to form ion channels in planar lipid bilayers was investigated in a wide spectrum of buffered pH ranging from 3.6 to 7.4. At pH 4.6, by supplementing the assay medium with 10 nM AnxVI in the cis chamber, we observed incorporation of AnxVI into the lipid bilayer within 3 min at 25°C. To achieve similar effects at pH 5.6, the protein concentration in the cis chamber was sixfold larger. The channel activity of AnxVI was inhibited by raising the pH of the assay medium to pH 7.4. Figure 1A shows typical recordings of the channel activity of AnxVI after its incorporation into asolectin bilayers under different holding electrode potentials. The frequencies and durations of the events permitted the construction of the amplitude distribution histograms. It appears that AnxVI is characterized by a single channel conductance oscillating between two states (open and closed), without any intermediate states. On the basis of such an analysis, it was possible to calculate the mean open and close times () for the AnxVI channels at different electrode potentials. At +30 mV potential, open amounted to 10 ± 0.1 ms and close to 10 ± 0.1 ms, whereas at an electrode potential of -20 mV the respective values were 4.0 ± 0.1 and 4.7 ± 0.1 ms. Therefore, the differences of varied with applied voltage, suggesting the probability that channel opening is voltage-dependent. A single channel conductance amounted to 23.4 ± 0.3 pS under symmetric conditions (50 mM/50 mM CsCl). The reversal potential of the current was 0 mV (Fig. 1B ). The selectivity of the AnxVI channel for transported ions was analyzed using a gradient of CsCl across lipid bilayers (cis/trans molar ratio was 1:4). Under these conditions, the reversal potential of the current was +10 mV. The calculated cation to anion permeability ratio amounted to 2, pointing to a very weak cation selectivity of the ion channels formed by AnxVI. Zn²⁺ and La³⁺, reported to be calcium channel inhibitors, did not affect the AnxVI channel kinetics in the concentration range from 1 to 10 mM.

View larger version (19K): [in this window] [in a new window]   Figure 1. Ion channel properties of AnxVI. A) Single channel recordings of AnxVI incorporated into lipid membranes. The planar phospholipid bilayers were formed in a 250 µm diameter hole drilled in a Derlin partition, separating two chambers cis and trans of 3 and 4 ml internal volume, respectively. Both chambers contained 10 mM citrate buffer, pH 4.5, 0.1 mM EGTA, 50 mM CsCl. The membranes were formed with a painted asolectin solution in decane (20 mg/ml). Current changes were induced by the addition of 10 nM AnxVI into the cis chamber and recorded at different holding potentials from 30 mV to -30 mV. Amplitude histograms were measured under the following conditions: electrode potential +30 mV, mean open channel amplitude 0.68 pA, and bin size of 0.01 pA. B) The current-voltage relationship for the open AnxVI channel. Filled circles correspond to an experiment carried out under symmetric conditions, i.e., cis and trans chambers contained 10 mM citrate buffer, pH 4.5, 0.1 mM EGTA, and 50 mM CsCl. The slope obtained from the linear fit corresponds to a single channel conductance of 24.3 ± 0.3 pS. Open squares correspond to asymmetric conditions: cis chamber contained 50 mM, whereas trans chamber contained 200 mM CsCl. The reversal potential for a single channel current amounted to +10 mV. The channel ion selectivity, expressed in terms of the cation to anion permeability ratio, amounted to 2, suggesting a weak cation selectivity. The results represent at least 3 determinations, which varied by 5%.

3. The molecular mechanism of ion channel activity of AnxVI at low pH is based on changes of protein hydrophobicity and on conformational transitions, namely, the appearance of new ß-sheet structures
Potential changes of AnxVI hydrophobicity induced by pH were measured with the use of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). TNS is a fluorescence probe that shows weak fluorescence in aqueous solution. In hydrophobic environments, its fluorescence intensity becomes enhanced by severalfold. The fluorescence of TNS in the presence of AnxVI increased at lower pH. At pH 4.6, it was approximately 20-fold higher than in the pH range from 7.0 to 8.3. These results can be interpreted as an exposure of hydrophobic surfaces by AnxVI at pH below 6.0, consistent with the binding of AnxVI to asolectin liposomes at acidic pH. The enhancement of TNS fluorescence in the presence of AnxVI at acidic pH was accompanied by fluorescence energy transfer from the AnxVI Trp residues to TNS. These results suggested changes in the Trp environment were induced by lower pH. This interpretation is further corroborated by a red shift of AnxVI intrinsic fluorescence maximum (by 12 nm) with decreasing pH. The exposure of hydrophobic domains within the AnxVI molecule could be related either to the rearrangement of secondary structure in AnxVI or to the protonation of side chain groups of the protein, making them more hydrophobic. Therefore, we examined the secondary structure of AnxVI using two methods: circular dichroism and infrared spectroscopy. The CD spectrum of AnxVI at pH 7.4 is characteristic for a protein with prevailing -helix content (80 ± 2%). This is consistent with the crystal structure of AnxVI. Lowering the pH from pH 7.4 to pH 3.0 resulted in a decrease of -helix content, the appearance of new ß structures (23 ± 2%) and an increase in the number of ß -turn segments within the AnxVI molecule compared to pH 7.4. Similar changes were observed at pH 6.2 (although the increase of ß structures was smaller: 8.4 ± 1.0%). The structural changes described in solution were almost fully reversible upon alkalinization of the assay medium. To probe the effects of pH on the secondary structure of AnxVI, infrared spectra of the protein in buffers prepared from ²H₂ O were also measured at p²H 7.1 and at p²H 4.7. The infrared spectrum in the amide-1 region of AnxVI under neutral conditions indicated a broad band centered at 1652 cm^-1. Upon mild acidification of an annexin sample to p²H 4.7, a new component band appeared at around 1632 cm^-1, characteristic of ß-sheet structures, confirming the observations made by CD spectroscopy.

CONCLUSIONS

The existence of a Ca²⁺-independent fraction of AnxVI in the cell has previously been reported, pointing to the idea that the mechanism of interaction of AnxVI with membranes is rather complex. In this work, we provide for the first time experimental evidence that Ca²⁺-independent mechanisms may involve local changes of intracellular pH allowing the binding of AnxVI to membranes at low Ca²⁺ concentrations existing in nonstimulated cells. It was also reported that AnxVI molecules may behave like membrane integral proteins: they are resistant to chelators and can be extracted from the membranes only by detergents. Such behavior may suggest that there is a mechanism allowing not only Ca²⁺-independent binding of AnxVI to membranes, but also its penetration into the membrane hydrophobic core. Indeed, we found that AnxVI binds to artificial lipid membranes in a Ca²⁺-independent, low pH-stimulated manner and behaves as a membrane integral protein. The binding is a prerequisite for the formation of ion channels by AnxVI molecule at acidic pH. However, the molecular mechanism leading to the formation of these channels by AnxVI at neutral pH remains obscure. Many in vitro observations rather are pointing to the surface character of the interaction of AnxVI with membranes: 1) concerning phospholipid specificity, annexins recognize the head portion of phospholipids rather than acyl chain length and conformation; 2) Ca²⁺-dependent AnxVI binding to membranes is sensitive to ionic strength and calcium chelating agents; 3) AnxVI at neutral pH exposes large hydrophilic surfaces to the milieu with many charged residues facing outside; 4) -helices within the AnxVI molecule are too short to allow complete protrusion of the protein within the membrane thickness; 5) AnxVI does not contain any known sequences targeting it to intracellular membrane compartments. We did not measure any AnxVI ion channel activity at neutral pH either in the presence or absence of Ca²⁺ even though AnxVI binds to membranes, especially to those enriched with acidic phospholipids. In the course of the present study, we observed the formation of ion channels by AnxVI induced by low pH. These channels, although voltage-dependent with a similar single channel conductance as channels formed by other annexins at neutral pH, are not specific to transported ions. Furthermore, the formation of ion channels by AnxVI molecules is accompanied by the penetration of the protein into the hydrophobic core of the membrane bilayer, as evident from monolayer experiments and from profound changes in protein hydrophobicity at acidic pH.

During our investigations, we observed pH-induced effects on the secondary structure of AnxVI. One likely mechanism is the neutralization of charges, which renders the surface of the protein more hydrophobic, leading to the movement of domains by forming new ß-sheet structures. This structural change would minimize the exposure of the hydrophobic surface of AnxVI toward aqueous solutions. Alternatively, protein–protein interactions may favor intermolecular ß-sheet structures to minimize hydrophobic exposure to the solvent. The current model of our understanding of the mechanism of the channel formation by AnxVI at acidic pH is shown in Fig. 2 .

View larger version (50K): [in this window] [in a new window]   Figure 2. The current model of formation of AnxVI ion channels at acidic pH. At neutral pH, AnxVI binds to the membranes in a reversible, Ca2+-dependent manner. The binding is stimulated by acidic phospholipids, assured by the ability of AnxVI to recognize head portions of lipid molecules. The formation of ternary complexes of AnxVI-Ca2+-phospholipid is not accompanied by a significant change in the annexin conformation. The binding is sensitive to ionic strength and calcium chelators and is undertaken at the surface of the membrane, without significant insertion of the annexin into the membrane hydrophobic core. At mildly acidic pH, AnxVI undergoes profound conformational changes even in the absence of phospholipids that lead to changes in protein hydrophobicity, its solubility and molecular shape. These changes are a prerequisite for binding of AnxVI into the membrane in a Ca2+-independent manner, insertion of the protein into membrane hydrophobic region and formation of ion channels. The ion transport through these channels is inhibited by raising the pH to 7.4, although AnxVI does not dissociate from the membrane. A pH-induced interaction of AnxVI with the membrane is insensitive to the specific phospholipids and probably leads to the appearance of a detergent-soluble fraction of AnxVI, behaving like a membrane integral protein.

Further investigations will need to establish whether such an activity of AnxVI reflects any possible in vivo function of this abundant protein. In this respect, participation of AnxVI in the regulation and maintenance of Ca²⁺ homeostasis in cardiomyocytes, as shown by transgenic animal models, is one likely possibility, especially under pathogenic conditions such as cardiac ischemia, which is characterized by a drop in intracellular pH below 6.5. One may also consider the fact that the pH value at the membrane surface is lower by 1.6 pH units in comparison to the cytosol, allowing penetration of AnxVI into membrane hydrophobic core.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0523fje ; to cite this article, use FASEB J. (February 26, 2001) 10.1096/fj.00-0523fje

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