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Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton, United Kingdom
Correspondence: Address reprint requests to Prof. A. G. Lee, Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK. Tel: 44-23-8059-4331; Fax: 44-23-8059-4459; E-mail: agl{at}soton.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). KcsA is a homotetramer, each monomer containing two transmembrane 
-helices separated by a conserved region, the P-loop that forms part of the ion conduction pathway. The pore structure of KcsA is believed to be very similar to those of the mammalian Kv and Kir family of K+ channels, and to those of the voltage-gated Na+ and Ca2+ channels (Sansom et al., 2002, Jiang et al., 2003). The presence of anionic phospholipid is essential for the function of KcsA, the channel remaining closed in the absence of anionic lipid (Heginbotham et al., 1998; Valiyaveetil et al., 2002). KcsA purified using dodecylmaltoside as detergent contains 
0.7 molecules of the anionic lipid phosphatidylglycerol per KcsA monomer and the crystal structure of KcsA shows one lipid molecule per monomer bound in a deep cleft at each monomer-monomer interface in the tetrameric structure (Fig. 1). The headgroup of this bound lipid molecule is not resolved in the crystal structure, and the lipid has therefore been modeled as a diacylglycerol with one C14 and one C9 chain (Zhou et al., 2001), but it is presumed to be phosphatidylglycerol (Valiyaveetil et al., 2002). Anionic lipid is not required for the formation of the KcsA tetramer since tetramers are formed when KcsA is reconstituted into bilayers of zwitterionic phospholipid in the absence of anionic lipid (Valiyaveetil et al., 2002; Williamson et al., 2002). Instead, it has been suggested that the presence of the anionic lipid "co-factor" bound between the transmembrane -helices is important in gating of the channel because opening and closing the gate must involve movement of the transmembrane -helices, which will be affected by the presence of the lipid molecule bound between the transmembrane -helices (Valiyaveetil et al., 2002).
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; Harrenga and Michel, 1999; McAuley et al., 1999; Lange et al., 2001; Camara-Artigas et al., 2002). These lipid molecules are often essential for activity, with cardiolipin, for example, being required for activity of many of the proteins important in bioenergetics (Jiang et al., 2000; Schlame et al., 2000; Heimpel et al., 2001). Binding sites for hydrophobic molecules located between transmembrane -helices have been referred to as non-annular sites, to distinguish them from the boundary or annular sites that make up the lipid-exposed external surface of the protein (Simmonds et al., 1982). The idea of non-annular binding sites developed from fluorescence quenching studies with the Ca2+-ATPase of sarcoplasmic reticulum, that showed the existence of sites on Ca2+-ATPase to which hydrophobic molecules could bind, but from which normal phospholipids were excluded (Simmonds et al., 1982; de Foresta et al., 1989; Lee, 2002).
Binding constants for phospholipids at the annular sites around a variety of membrane proteins have been determined (Marsh and Horvath, 1998; Lee, 2003) but there have as yet been no measurements for binding constants of phospholipids at non-annular sites. It is not known to what extent binding at non-annular sites is structurally specific, and it is not known if binding of anionic phospholipids to these sites is driven by nonspecific charge effects or by specific interactions such as hydrogen bonding. Further, the lack of knowledge about the binding constants at these sites means that it is not known which lipids will occupy these sites in the native membrane and whether or not these sites will be fully occupied in the native membrane.
In previous studies we have shown that lipid binding constants for the annular sites around KcsA can be obtained using fluorescence quenching methods in which the degree of occupancy of the lipid binding sites around KcsA by bromine-containing phospholipids is determined from the level of quenching of the fluorescence of the Trp residues in the protein (Williamson et al., 2002). Phospholipids containing brominated fatty acyl chains are prepared by addition of bromine across the double bond in the corresponding phospholipid containing cis-unsaturated fatty acyl chains. Phospholipids containing brominated fatty acyl chains behave much like conventional phospholipids with unsaturated fatty acyl chains, because the bulky bromine atoms have effects on lipid packing that are similar to those of a cis double bond (East and Lee, 1982). KcsA contains five Trp residues of which Trp-26 and Trp-113, at the cytoplasmic ends of transmembrane -helices M1 and M2 respectively, and Trp-87 at the extracellular end of M2, are exposed to the lipid bilayer. Binding of brominated phospholipids to the annular sites around KcsA would be expected to lead to quenching of the fluorescence of these lipid-exposed Trp residues. In contrast, Trp-67 and Trp-68 are located away from the lipid-protein interface as part of the short pore helix that points into the intracellular cavity. However, both Trp-67 and Trp-68 are close to the sn-1 chain of the lipid bound at the non-annular site, which is inserted between the pore helix of one monomer and a transmembrane -helix of the adjacent monomer (Fig. 1). Thus binding of brominated anionic phospholipids to the non-annular sites would also be expected to result in quenching of Trp fluorescence. Importantly, KcsA still forms tetramers in the absence of phosphatidylglycerol (Williamson et al., 2002; Valiyaveetil et al., 2002) so that binding to the non-annular binding sites can be treated as a simple equilibrium binding reaction. Here it is shown that binding constants at the non-annular sites can indeed be obtained from analysis of fluorescence quenching data.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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to give dibromostearoylphosphatidylcholine (BrPC), dibromostearoylphosphatidylethanolamine (BrPE), dibromostearoylphosphatidic acid (BrPA), dibromostearoylphosphatidylserine (BrPS), dibromostearoylphosphatidylglycerol (BrPG), and dibromostearoylcardiolipin (BrCL), respectively.
Purification of KcsA and reconstitution
A plasmid containing the kcsA gene (Schrempf et al., 1995) with a poly-His epitope at the N-terminus was the generous gift of Professor Schrempf. KcsA was purified using Mega-9 (Calbiochem, Nottingham, UK) as detergent, as described in Williamson et al. (2002). Purified KcsA was reconstituted into lipid bilayers by mixing lipid and KcsA in cholate, followed by dilution into buffer to decrease the concentration of cholate below its critical micelle concentration, as described (Williamson et al., 2002). For reconstitution into bilayers of a single phospholipid, the required phospholipid (0.6 µmol) was dried from a chloroform solution onto the walls of a thin glass vial. Buffer (300 µl; 20 mM HEPES and 1 mM EGTA at pH 7.2) containing 20 mM cholate was added and the sample was sonicated to clarity in a bath sonicator (Ultrawave, Cardiff, UK). KcsA (100 µg) was then added and the suspension left at room temperature for 15 min, followed by incubation on ice until use. For reconstitution into bilayers containing a mixture of two lipids, stock solutions of the two lipids (2 mM lipid) were prepared in cholate-containing buffer as described above. Aliquots of the two stock solutions were then mixed in the appropriate proportions to give 200-µl samples of the mixed lipids. The mixtures were incubated at 60°C for 15 min and then left at room temperature for 1 h and then mixed with KcsA, as described above. For reconstitutions with phosphatidylethanolamine, this procedure sometimes gave inconsistent results; and in these cases lipid mixtures were first prepared by mixing the lipids in chloroform solution, which were then dried down and dissolved in cholate, followed by reconstitution as described above.
Fluorescence measurements
For fluorescence measurements 50 µl of the sample were diluted into 3 ml buffer (20 mM HEPES and 1 mM EGTA, at pH 7.2) and the fluorescence recorded on an SLM (Urbana, IL) 8000C fluorimeter with excitation at 290 nm, at 25°C. Unless otherwise stated, the concentration of KcsA was 0.24 µM and the molar ratio of lipid to KcsA was 100:1. Wavelengths of maximum fluorescence emission intensity were obtained by fitting fluorescence emission spectra to skewed Gaussian curves, as described in Williamson et al. (2002).
Analysis of fluorescence results
Quenching of Trp fluorescence by bromine-containing molecules requires the Trp residue and the bromine atom to be close in space. Quenching of the fluorescence of a particular Trp residue in KcsA by a brominated phospholipid molecule will therefore depend on the level of occupancy of the lipid binding sites immediately adjacent to that Trp residue by brominated phospholipid. Quenching of Trp fluorescence due to binding of brominated phospholipids to the annular sites around KcsA has been fitted to a lattice model for quenching (London and Feigenson, 1981; Caffrey and Feigenson, 1981; Mall et al., 1998; O'Keeffe et al., 2000) using the equation
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The experimental data were also fitted to a model involving separate annular and non-annular binding sites, as in the previous analysis of binding at non-annular sites on the Ca2+-ATPase (Simmonds et al., 1982). It is assumed that, although both anionic and zwitterionic lipids can bind to the annular sites, possibly with different affinities, only anionic phospholipids can bind to the non-annular sites. The degree of occupation of the non-annular sites, 
, by brominated anionic phospholipid, is given by
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)
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). The fluorescence emission maxima for KcsA reconstituted into bilayers of anionic lipid are identical to that in DOPC (Fig. 2; Table 1). Spectra were uncorrected and differ slightly from those reported in Williamson et al. (2002), which were recorded on a different instrument. The fluorescence intensity in DOPA is 10% lower than that in DOPC (Fig. 2). In mixtures of DOPC and DOPA or DOPC and DOPS fluorescence intensities decrease with increasing mole fraction of anionic lipid, the maximum fluorescence quenching observed in DOPS being the same as that in DOPA (Figs. 3 and 4). The presence of DOPG (Fig. 2) or TOCL resulted in no significant decrease in fluorescence intensity. Fluorescence intensities for KcsA reconstituted into DOPE are more variable between reconstitutions than those with other phospholipids, possibly connected with the tendency of DOPE to form nonlamellar phases, but the fluorescence emission maximum was very similar to that in the other phospholipids (Table 1), and the fluorescence intensity in a 9:1 mixture of DOPE:DOPC, in which intensities were reproducible, were the same as in DOPC.
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). Quenching in brominated phosphatidylethanolamine and phosphatidylglycerol fits to an n number the same as for phosphatidylcholine (Table 1). Quenching by brominated cardiolipin fits to a value for n of 0.90 ± 0.15 (Fig. 5), a value close to half that for the other phospholipids, consistent with the four-chain structure of cardiolipin compared to the two-chain structure of the other phospholipids. Quenching in brominated phosphatidic acid fits to a significantly larger n number of 2.49 ± 0.25 (Fig. 5), possibly because of the small size of the phosphatidic acid headgroup. For the subsequent analyses, an n-value of 1.69 was used for all two-chain phospholipids except for phosphatidic acid, where an n-value of 2.49 was used. In studies of quenching in mixtures of cardiolipin and phosphatidylcholine, the mole fraction of cardiolipin was calculated based on the number of moles of fatty acyl chains to account for the fact that TOCL contains four chains and DOPC contains two chains; an n-value of 1.69 was used to describe quenching.
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10% caused by the lipid headgroups of phosphatidic acid and phosphatidylserine. The level of quenching observed with BrPS decreases with increasing ionic strength; in 500 mM KCl, the level of quenching (0.60 ± 0.01; Fig. 6) is comparable to that in BrPC.
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). Adapting this same model to KcsA, fluorescence quenching for KcsA can be described by Eq. 5 where both phosphatidylserine and phosphatidylcholine can bind to the annular sites but only anionic lipid can bind to the non-annular sites. The model further assumes that binding to both the annular and the non-annular sites can quench the fluorescence of all the Trp residues; as with the previous experiments with Ca2+-ATPase it is assumed that the level of quenching following from binding to the non-annular sites is directly proportional to the occupancy of the sites by BrPS. As shown in Fig. 3 B, the data for quenching in BrPS/DOPC mixtures fits well to Eq. 5, with a relative binding constant of 0.83 for BrPS relative to DOPC at the annular sites and a binding constant of 2.53 ± 0.32 mole fraction-1 for binding at the non-annular sites (Table 3).
Quenching of Trp fluorescence by brominated phospholipids requires the brominated lipid and Trp to be close, quenching fitting to a Förster-type equation with a value of Ro—the distance at which quenching is 50% efficient—of 8–9 Å (Bolen and Holloway, 1990; Mall et al., 2001). Three of the Trp residues in KcsA (Trp-87, Trp-67, and Trp-68) are on the same side of the membrane as the lipid molecule bound to the non-annular site, and close to it; the other two Trp residues (Trp-26 and Trp-113) are more distant, on the opposite side of the membrane (Valiyaveetil et al., 2002). The data were also therefore fitted to a model (Eq. 6) in which only 60% of the Trp fluorescence could be quenched from the non-annular sites. This also gave a good fit to the data, with an increased value for the non-annular binding constant of 5.71 ± 0.32 mole fraction-1 (Fig. 3 B, Table 3).
Fits of fluorescence quenching data for BrPC/DOPS and BrPS/DOPC mixtures in the presence of 500 mM KCl to Eq. 2 gave binding constants much closer than those obtained at low salt, although the binding constant DOPS relative to DOPC derived from the BrPS/DOPC experiments was still slightly greater than that derived from the BrPC/DOPS experiments (Fig. 6, Table 2). This suggests that the non-annular binding constant of BrPS is reduced at high ionic strength. Thus fits of the data to the two-site, annular/non-annular binding site models (Eqs. 5 and 6) give much reduced binding constants for the non-annular site compared to those obtained at low salt (Fig. 6, Table 3).
Fluorescence quenching in mixtures with phosphatidic acid (Fig. 4) and phosphatidylglycerol (Fig. 8) are very similar to those in mixtures of phosphatidylserine. Quenching is again more marked at low mole fractions of brominated lipid when the brominated lipid is the anionic lipid than when it is BrPC. The data fit to Eq. 2 with the parameters listed in Table 2 and to Eqs. 5 and 6 with the parameters listed in Table 3. Effects of 500 mM KCl on quenching in mixtures with phosphatidic acid are less marked than those on quenching in mixtures with phosphatidylserine (Fig. 4). Fluorescence quenching in mixtures with cardiolipin (Fig. 9) are consistent with relatively weak binding of cardiolipin at the annular sites with strong binding at the non-annular sites (Tables 2 and 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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; Valiyaveetil et al., 2002). Anionic lipid is not required for formation of the KcsA tetramer, since tetramers are formed when KcsA denatured in SDS is reconstituted into bilayers of phosphatidylcholine (Valiyaveetil et al., 2002). Rather, it was suggested that the anionic phospholipid bound between the transmembrane -helices of the channel affected channel opening because channel opening involves movement of the transmembrane -helices (Valiyaveetil et al., 2002). KcsA purified from E. coli membranes using dodecylmaltoside as detergent contains 0.7 mole fraction of phosphatidylglycerol per mole of KcsA monomer, suggesting the presence of a single site per monomer with relatively high affinity for phosphatidylglycerol; it is assumed that it is binding to this site that affects channel opening (Valiyaveetil et al., 2002). The crystal structure of KcsA shows a single lipid molecule per monomer, modeled as a diacylglycerol because the headgroup structure was not resolvable, but assumed to be a phosphatidylglycerol molecule, bound in a deep cleft (Zhou et al., 2001; Valiyaveetil et al., 2002), in a type of site referred to previously as a non-annular binding site (Simmonds et al., 1982; Lee, 2003). The crystal structure also shows a single chain modeled as nonan-1-ol, probably corresponding to part of the chain of a detergent molecule. The chain is located in a shallow groove on the surface of the protein, illustrating how the annular phospholipids might bind to the hydrophobic surface of the protein (Fig. 1).
The Trp residues in KcsA are located in two bands, one on each side of the membrane close to the expected interface between the hydrocarbon core of the bilayer and the lipid headgroup region (Williamson et al., 2002). The observation that reconstitution into bilayers of anionic phospholipid results in no significant change in the position of the fluorescence emission maximum (Table 1) suggests that no large conformation change for KcsA occurs on reconstitution into anionic phospholipid. Fluorescence intensities in DOPS and DOPA are 10% lower than in DOPC (Table 1). Trp fluorescence is quenched in solution by acidic groups, by a mechanism involving electron or proton transfer requiring close contact between the Trp and the acidic group; oleic acid, for example, has been shown to quench the Trp fluorescence of Ca2+-ATPase and of a simple hydrophobic analogue of Trp incorporated into lipid bilayers (Froud et al., 1986). Quenching of the Trp fluorescence of KcsA by DOPS and DOPA is consistent with a location for the lipid headgroups close to some or all of the Trp residues on KcsA.
Reconstitution of KcsA into bilayers of lipids containing 9,10-dibromostearyl chains leads to extensive quenching of fluorescence (Table 1). Levels of fluorescence quenching in bilayers of the brominated anionic phospholipids, when corrected for the effect of the anionic lipid headgroup, are very similar to that observed in BrPC (Table 1). Since the level of fluorescence quenching of KcsA observed in bilayers of brominated phospholipid depends on the distance of separation between the bromine groups and the Trp residues (Williamson et al., 2002) this observation again argues against any large change in conformation for KcsA on reconstitution into bilayers of anionic phospholipid.
Lipid binding constants
Relative lipid binding constants for the annular sites around membrane proteins have been estimated from electron spin resonance (ESR) and fluorescence studies (Marsh and Horvath, 1998; Williamson et al., 2002; Lee, 2003). However, there have, as yet, been no reports of binding constants for phospholipids at non-annular sites. Here we have shown that such information can be obtained for KcsA from fluorescence quenching studies with brominated phospholipids. Fluorescence quenching in mixtures of BrPC with anionic phospholipids are consistent with simple competition between anionic phospholipids and BrPC for binding at the annular sites, with binding constants for the anionic phospholipids relative to DOPC close to 1 except for cardiolipin (Table 3), suggesting that charge interactions are relatively unimportant for binding to annular sites. Studies of binding of anionic phospholipids to a range of membrane proteins using ESR methods also showed little selectivity in binding, binding of the anionic phospholipids being no more than a factor of two stronger than phosphatidylcholine (Marsh and Horvath, 1998). Binding of phosphatidylethanolamine to the annular sites on KcsA is slightly weaker than binding of phosphatidylcholine (Table 2), despite the fact that phosphatidylcholine is absent from the cytoplasmic membrane of Streptomyces lividans whose major zwitterionic lipid is phosphatidylethanolamine (Hoischen et al., 1997). Phosphatidylethanolamine was also found to bind less strongly than phosphatidylcholine to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, a membrane in which phosphatidylcholine is the major zwitterionic lipid (East and Lee, 1982). The binding constant for phosphatidylethanolamine is similar to that for cardiolipin (Table 3) and it is possible that this is related to the fact that both these phospholipids have a tendency to form curved, hexagonal HII phases as well as the normal planar bilayer phase.
Quenching in mixtures of brominated anionic phospholipid with DOPC was more marked at low mole fractions of brominated phospholipid than would have been expected if binding at the annular sites showed little specificity for anionic phospholipids compared to DOPC (Figs. 3, 4 and 8). This is consistent with binding of the brominated anionic phospholipids to the non-annular binding sites for anionic phospholipids observed in the crystallographic studies of KcsA (Fig. 1), sites at which zwitterionic phospholipids can either not bind, or to which they bind with very low affinity. Quenching by brominated anionic phospholipids will then arise both from binding to the annular sites and from binding to the non-annular sites. A model describing quenching of this type was proposed previously in studies of the binding of brominated cholesterol to Ca2+-ATPase (Simmonds et al., 1982), and is used here. An unknown in the fitting procedure is the fraction of the Trp fluorescence that can be quenched from the non-annular sites. Inasmuch as quenching of Trp fluorescence by bromine requires the bromine to be located close to the Trp residue (Bolen and Holloway, 1990; Mall et al., 2001), and inasmuch as three of the five Trp residues in KcsA are on the same side of the membrane as the non-annular site, it is probable that the fluorescence of only 60% of the Trp residues will be quenched from the non-annular sites (Eq. 6) and all the data reported here fit to this model (Table 3). However, all the data also fit to a model in which binding at the non-annular sites quenches the fluorescence of all the Trp residues (Eq. 5). It is not possible to distinguish between these two possibilities from the data presented here, but the experiments do provide information about the likely order of magnitude of the binding constants and about their dependence on lipid headgroup structure.
Non-annular binding constants for phosphatidylserine and phosphatidic acid are equal within experimental error, the non-annular binding constant for phosphatidylglycerol being significantly lower and that for cardiolipin being significantly higher (Table 3). The binding constant for phosphatidylserine decreases very markedly in the presence of 500 mM KCl (Table 3), suggesting that binding at the non-annular binding sites is strongly dependent on electrostatic interactions. The effect of 500 mM KCl on the non-annular binding constant for phosphatidic acid is less marked than its effect for phosphatidylserine (Fig. 4; Table 3) suggesting that charge interactions make less of a contribution to binding of phosphatidic acid than phosphatidylserine. Weaker binding of phosphatidylglycerol than of the other anionic phospholipids (Table 3) could be a result of the presence of the relatively bulky and hydrophobic glycerol moiety in the lipid headgroup. The anionic phospholipid headgroups probably interact with Arg-64 and Arg-89 located in the girdle of charged residues above Trp-87, the interaction being a relatively nonspecific one since the anionic lipid headgroup is not resolved in the crystal structure (Fig. 1).
Binding of anionic lipids to the non-annular binding site in the native membrane
The plasma membrane of the gram-negative Escherichia coli in which KcsA is expressed contains 20% anionic phospholipid, predominantly phosphatidylglycerol; it is not known if the distribution of phosphatidylglycerol between the two leaflets of the bilayer is uniform or asymmetric (Huijbregts et al., 2000). If the binding constant for phosphatidylglycerol is 1.46 mole fraction-1 (Table 3) then the non-annular binding site would be 25% occupied at a mole fraction of phosphatidylglycerol of 0.2 and it would be 40% occupied at a mole fraction of phosphatidylglycerol of 0.4, corresponding to the situation where all the phosphatidylglycerol was in the outer leaflet of the plasma membrane, the side on which the non-annular binding site of KcsA is located. Fractional occupation of the non-annular binding site by anionic phospholipid in the native membrane would be surprising, inasmuch as the site needs to be occupied for KcsA to be functional (Valiyaveetil et al., 2002). However, the lipid composition of the plasma membrane of the Gram-positive S. lividans is different from that of KcsA. The lipid composition of S. lividans appears not to have been determined, but in S. ambofaciens 40% of the lipid is cardiolipin with small amounts of phosphatidylinositol as the only other anionic lipid (Schauner et al., 1999) and in S. hygroscopicus 20% of the lipid is cardiolipin with 5% phosphatidic acid and 10% modified phosphatidylinositols (Hoischen et al., 1997). The phosphatidylglycerol content of the Streptomyces family appears to vary widely between strains (Lechevalier et al., 1981). Given the strong binding of cardiolipin to the non-annular binding site in KcsA (Table 3) it is likely that the site will be occupied by cardiolipin in the native membrane. With a binding constant of 7.65 mole fraction-1 for cardiolipin expressed on a chain basis, the non-annular site would be 75% occupied in a membrane containing 20% cardiolipin and 90% occupied in a membrane containing 40% cardiolipin.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Submitted on February 4, 2003; accepted for publication August 25, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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), phosphatidylserine; (
), phosphatidic acid; and (
), cardiolipin. The lines show fits to 
), BrPA and DOPC (
