extfieldsofstudy: Chemistry Medicine

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Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing.

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca2+-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca2+ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu51, Glu55, or Glu58, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp59, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca2+ dissociation toward the cytoplasmic side. Mutation of Leu60 to alanine or proline and of Val62 to alanine also enhanced Ca2+ dissociation, whereas an 11-fold reduction of the rate of Ca2+ dissociation was observed upon alanine substitution of Leu65, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca2+ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp59, and alanine substitution of Leu65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca2+ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca 2؉ -ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca 2؉ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu 51 , Glu 55 , or Glu 58 , located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp 59 , located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca 2؉ dissociation toward the cytoplasmic side. Mutation of Leu 60 to alanine or proline and of Val 62 to alanine also enhanced Ca 2؉ dissociation, whereas an 11-fold reduction of the rate of Ca 2؉ dissociation was observed upon alanine substitution of Leu 65 , thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca 2؉ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp 59 , and alanine substitution of Leu 65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca 2؉ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.
The Ca 2ϩ -ATPase 1 of sarcoplasmic reticulum is a membrane-bound enzyme that pumps Ca 2ϩ from the cytosol to the lumen of the sarcoplasmic reticulum at the expense of chemical energy being released by ATP hydrolysis (1)(2)(3)(4). It belongs to the family of P-type ATPases characterized by the formation during the enzymatic cycle of a phosphorylated enzyme intermediate, in which the ␥-phosphoryl group of ATP has been transferred to a conserved aspartic acid side chain in the enzyme. The phosphoenzyme intermediate is fundamental to the coupling between the spatially well separated processes of ATP hydrolysis in the cytoplasmic part of the molecule and vectorial ion transport across the membrane. Phosphorylation of the enzyme by ATP is activated by Ca 2ϩ binding at cytoplasmically facing high affinity sites of the E 1 form. During the subsequent conformational change, [Ca 2 ]E 1 P 3 Ca 2 E 2 P, the Ca 2ϩ binding sites are reoriented toward the lumen and their affinity markedly lowered, resulting in release of the bound ions (Scheme 1). It is believed that, following their binding at the high affinity sites, the Ca 2ϩ ions are contained within an "occluded" state (here indicated by brackets around the Ca 2ϩ ions) with no access to the medium on either side of the membrane (5)(6)(7).
The overall tertiary structure of the Ca 2ϩ -ATPase comprises 10 transmembrane ␣-helices (M1-M10) and a large cytoplasmic headpiece divided into three distinct subdomains denoted "actuator" (A), "nucleotide-binding" (N), and "phosphorylation" (P) domain (8,9). The two Ca 2ϩ binding sites, located in the membranous part of the molecule more than 40 Å away from the cytoplasmic catalytic site, are formed by amino acid residues in transmembrane segments M4 (Val 304 , Ala 305 , Ile 307 , and Glu 309 ), M5 (Asn 768 and Glu 771 ), M6 (Asn 796 , Thr 799 , and Asp 800 ), and M8 (Glu 908 ) (8, 10 -12). Although the nature of the intramolecular signaling between the distantly located catalytic site and the Ca 2ϩ binding sites in the membrane is yet incompletely understood, the propagation of conformational changes seems to be part of this long range communication. The crystal structure has been determined for two forms of the Ca 2ϩ -ATPase, a Ca 2ϩ -bound form presumably corresponding to [Ca 2 ]E 1 (8) and a Ca 2ϩ -free form with thapsigargin bound, believed to resemble E 2 ("E 2 -Tg") (9). Comparison of the two structures reveals major conformational differences. In particular, the A-, N-, and P-domains move from a spatially well separated organization in the Ca 2ϩ -bound form to a more compact entity in E 2 -Tg (9). Interestingly, it appears that this rearrangement of the cytoplasmic domains is accompanied by a partial unfolding and bending (at Asp 59 ) of the helical structure of transmembrane segment M1, to which the A-domain is physically connected through a flexible hinge. The bending may be aided by the amphipathic nature of the most N-terminal part of M1, allowing one side of the bent part to remain associated with membrane lipid while the other side comes into contact with the cytoplasmic phase (9). An important challenge is to reveal whether these rearrangements of the cytoplasmic domains and M1 are coupled and occur in the native state during the transport cycle and to understand the functional implications. Although it was recently reported that the length and, hence, the flexibility of the hinge linking M1 and the A-domain is critical to phosphoenzyme processing (i.e. Reactions 4 -6 in Scheme 1) (13), the functional importance of M1 has so far not been characterized in any detail. A most striking aspect of M1 of the SERCA family of Ca 2ϩ -ATPases is the presence of four negatively charged amino acid residues (Glu 51 , Glu 55 , Glu 58 , and Asp 59 ), which have been suggested to provide the cytoplasmic entry pathway to the membranous Ca 2ϩ sites (14). This proposal was mainly based on the crystal structure of the Ca 2ϩ -ATPase in the Ca 2ϩ -bound form (8), but, since then, the determination of the crystal structure of the Ca 2ϩ -ATPase in the E 2 -Tg form has attracted further attention to M1 by demonstrating that the above-described bending of M1 opens a water-accessible channel leading between M1 and M3 to Glu 309 at Ca 2ϩ site II, a channel with the potential of a Ca 2ϩ entry port (9). Of Glu 51 , Glu 55 , Glu 58 , and Asp 59 in M1, the first three contribute to define the polar/charged surface of the amphipathic part near its N terminus, whereas Asp 59 is located right at the bend formed in the E 2 form. Furthermore, in the Ca 2ϩ -bound crystal structure, Glu 58 appears to be hydrogenbonded to Glu 309 at Ca 2ϩ binding site II, making a direct conformational effect on M1 possible upon Ca 2ϩ occupancy of the binding sites (14).
With respect to the specific Ca 2ϩ selectivity of the Ca 2ϩ -ATPase, it is interesting to note that the negatively charged M1 residues Glu 55 and Glu 58 are replaced by residues with positively charged side chains in the Na ϩ ,K ϩ -ATPase. Because transmembrane segment M3 of both ATPases contains additional negatively charged residues, which appear to be located rather close to M1 in the three-dimensional structure, the situation could be somewhat similar to voltage-gated Ca 2ϩ and Na ϩ channels, where the cation selectivity filter is formed by four negatively charged side chains in the former and two negative plus one positive side chain in the latter (15,16). Thus, the above-mentioned charged residues within the cytoplasmic part of M1 of the Ca 2ϩ -ATPase could possibly contribute to a Ca 2ϩ selectivity filter.
These considerations on the possible function of M1 of the Ca 2ϩ -ATPase in Ca 2ϩ migration and in long range signal transmission have prompted a detailed study of the structural and functional aspects of M1. To test the above-mentioned hypothesis about Glu 51 , Glu 55 , Glu 58 , and Asp 59 as part of a possible Ca 2ϩ entry pathway and/or selectivity filter, we have mutated these amino acids individually to the neutral leucine and to the oppositely charged and rather bulky arginine, which would be expected to impose electrostatic and steric effects on Ca 2ϩ migration, if positioned near the migration pathway. Moreover, we have substituted the hydrophobic amino acids Phe 57 , Leu 60 , Val 62 , and Leu 65 to investigate the demands for bulkiness and hydrophobicity of these side chains. Phe 57 is located on the hydrophobic surface of the bent part of M1 in the E 2 -Tg structure and could be instrumental in attaching this part to the lipid bilayer. The other hydrophobic residues are embedded in the membrane, Leu 65 below the level of the Ca 2ϩ ions in the Ca 2ϩ -bound structure. The effects of these mutations on the various partial reaction steps involved in Ca 2ϩ binding and catalysis have been analyzed, using among other assays also transient kinetic methods that allow measurement of the rates of conformational changes and Ca 2ϩ dissociation toward the cytoplasmic side.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis and Expression-The QuikChange sitedirected mutagenesis kit (Stratagene) was used to introduce the desired mutations directly into full-length cDNA encoding the rabbit fast twitch muscle Ca 2ϩ -ATPase (SERCA1a isoform). The template in the mutagenesis reaction consisted of SERCA1a cDNA inserted into the expression vector pMT2 (17). For expression of recombinant protein, pMT2 containing wild-type or mutant cDNA was transfected into mammalian COS-1 cells using the calcium phosphate precipitation method (18). The microsomal fraction containing expressed wild-type or mutant Ca 2ϩ -ATPase was isolated by differential centrifugation (19), and the expression level was quantitated by a specific enzyme-linked immunosorbent assay (20).
Functional Studies-The ability of wild-type or mutant Ca 2ϩ -ATPase to transport Ca 2ϩ and hydrolyze ATP was determined by studying the 45 Ca 2ϩ uptake into microsomal membranes and the P i liberation, respectively (21). The molecular turnover rate was calculated as the ratio between the specific Ca 2ϩ -ATPase activity and the active-site concentration. The latter was determined by quantitation of phosphorylation from [␥-32 P]ATP at 0°C in the presence of a saturating Ca 2ϩ concentration. Samples of wild-type and mutant Ca 2ϩ -ATPase were examined in parallel, and the molecular turnover rate corresponding to the mutants was expressed relative to that of the wild type. Partial reactions were studied according to previously described principles (20 -23), taking advantage of the acid stability of the phosphoenzyme formed from [␥-32 P]ATP or 32 P i , and details are given in the figure legends. A Bio-Logic quenched flow module QFM-5 or SFM-400/Q (Bio-Logic Science Instruments, Claix, France) was used for rapid mixing in experiments where the reaction time was below 1 s, applying the previously described protocols (22,23). In all phosphorylation experiments, the reaction was quenched by addition of 0.5-2 volumes of 25% (w/v) trichloroacetic acid containing 100 mM H 3 PO 4 . Subsequently, the acidprecipitated enzyme was washed by centrifugation, dissolved in loading buffer and subjected to separation by SDS-PAGE at pH 6.0 (24). The distribution of radioactivity associated with the gel was quantitated by electronic autoradiography of the dried gel using a Packard Cyclone TM Storage Phosphor System.
Data Analysis-Using the Sigmaplot program (SPSS Inc.), time courses of phosphorylation and dephosphorylation were fitted according to first-order kinetics, whereas ligand concentration dependences were fitted by use of the Hill equation. Generally, the experiments were performed at least twice on different enzyme preparations, and average values are shown in the figures. Standard errors are shown in the tables except for a few cases, in which the experiment (giving a wild type-like result) was performed only once.

Site-directed Mutagenesis and Expression-Several
Ca 2ϩ -ATPase point mutants were designed to study the functional importance of transmembrane segment M1. An overview of these mutations is given in Table I. Two different strategies were used: (i) charge reversal or removal (Glu 51 , Glu 55 , Glu 58 , and Asp 59 ) and (ii) hydrophobic size reduction (Phe 57 , Leu 60 , Val 62 , and Leu 65 ). The carboxylic acid residues were replaced by the positively charged and rather bulky arginine and with leucine, removing the negative charge. Phe 57 , Leu 60 , Val 62 , and Leu 65 were replaced by the smaller hydrophobic alanine to test the importance of the bulkiness of these side chains. In addition, Phe 57 was replaced by the more polar glutamine, and Leu 60 by proline. The expression level of all these Ca 2ϩ -ATPase constructs in COS cells was rather similar to that of the wild type (data not shown).
Effect on Overall Reaction-To characterize the effect of the M1 mutations on the overall function of the Ca 2ϩ -ATPase, the ability to hydrolyze ATP was determined at 37°C in the pres-SCHEME 1. Ca 2؉ -ATPase reaction cycle. Simplified scheme illustrating the partial reactions of the Ca 2ϩ -ATPase reaction cycle. Occluded Ca 2ϩ ions are shown in brackets.
ence and absence of the calcium ionophore A23187. Data obtained in the presence of ionophore are shown in Table I. Mutant Leu 65 3 Ala displayed the most pronounced reduction of maximal ATPase activity (to 3% of wild type). The maximal ATPase activity of Asp 59 3 Leu was reduced to 18%. In contrast, the maximal ATPase activity was increased in Val 62 3 Ala relative to wild type (by 30%). Upon addition of the calcium ionophore, passive efflux of Ca 2ϩ from the vesicles is facilitated, thereby relieving "back inhibition" caused by accumulation of a high concentration of Ca 2ϩ inside the vesicles (21). In the absence of calcium ionophore, the ATPase activity of wild type and all mutants except Leu 65 3 Ala was 2-to 3-fold lower than in its presence (data not shown), indicating that the ability to pump Ca 2ϩ and the luminal sensitivity to Ca 2ϩ were preserved. In accordance with this conclusion, direct measurement of ATP-dependent 45 Ca 2ϩ transport at 37°C in the presence of oxalate to trap Ca 2ϩ in the lumen of the microsomal vesicles demonstrated transport activity in all mutants except Leu 65 3 Ala (Table I). The mutational effects on ATP hydrolysis and Ca 2ϩ transport are not sufficiently different to signify any change in the coupling between the catalytic site in the cytoplasmic domain and the Ca 2ϩ binding sites in the transmembrane part of the molecule. Although the 30% increase of ATPase activity seen for Val 62 3 Ala is not accompanied by an increase of the Ca 2ϩ transport rate, this relatively minor difference may well be accounted for by the higher Ca 2ϩ concentration present in the vesicles during the Ca 2ϩ transport assay, which through Ca 2ϩ binding at the luminal sites slows the turnover of the phosphoenzyme, thereby tending to mask any accelerating mutational effect. Having established the impact of the various mutations in M1 on the overall functional performance of the Ca 2ϩ -ATPase, we proceeded with a more detailed analysis of the partial reactions of the enzyme cycle.
Ca 2ϩ Dependence of Phosphorylation from [␥-32 P]ATP-The Ca 2ϩ -mediated activation of phosphorylation from ATP requires the binding of both calcium ions at the high affinity cytoplasmically facing sites (cf. Reactions 2 and 3 in Scheme 1).
Examination of the mutational effect on Ca 2ϩ titration of phosphorylation therefore provides information about the Ca 2ϩbinding properties. The Ca 2ϩ dependence of steady-state phosphorylation was studied at 0°C, where phosphoenzyme accumulation is favored. Fig. 1 shows the titration curves for some of the mutants, and Table I summarizes the data for all mutants. Substitution of Glu 51 , Glu 55 , or Glu 58 by either leucine or arginine had no significant effect on the apparent Ca 2ϩ affinity, as exemplified by Glu 58 3 Arg in Fig. 1 (cf. Fig.  1 and Table I). In contrast, arginine substitution of Asp 59 caused a 5-fold reduction of apparent Ca 2ϩ affinity (increase of K 0.5 ) compared with wild type, whereas the apparent affinity for Ca 2ϩ was unaffected by leucine or alanine substitution of this residue. These results are in accordance with a previous study in which the Ca 2ϩ dependence of Ca 2ϩ transport was found normal in a cluster mutant with alanine substitutions of Glu 55 , Gln 56 , Glu 58 , and Asp 59 (25). The mutants Phe 57 3 Ala, Leu 60 3 Ala/Pro, and Val 62 3 Ala, with alterations to hydrophobic residues, displayed moderate reductions of apparent Ca 2ϩ affinity (1.5-to 3-fold increase of K 0.5 ). On the contrary, Leu 65 3 Ala displayed higher affinity for Ca 2ϩ than wild type, the K 0.5 being reduced to 61% of the corresponding value for the wild type ( Fig. 1 and Table I).
Rate of Ca 2ϩ Dissociation toward the Cytoplasmic Side-To determine more directly the effects of the mutations on Ca 2ϩ binding, we examined the rate of Ca 2ϩ dissociation toward the cytoplasmic side from the [Ca 2 ]E 1 form at pH 6.0, 25°C (Fig. 2), using a previously described quenched flow technique (23). Mutant or wild-type enzyme pre-equilibrated with saturating amounts of Ca 2ϩ was treated with an excess of the Ca 2ϩ chelator EGTA for various time intervals prior to a 34-ms incubation with [␥-32 P]ATP testing the ability to phosphorylate. Because occupancy of the Ca 2ϩ binding sites is required to render the enzyme phosphorylatable from ATP, the time course of disappearance of the ability to phosphorylate reflects the dissociation of Ca 2ϩ . Because the enzyme is not phosphorylated at the time when Ca 2ϩ dissociates, the dissociation must occur  (21) at 37°C. The medium contained 50 mM TES/Tris (pH 7.0), 100 mM KCl, 5 mM ATP, 7 mM MgCl 2 , 1 M Ca 2ϩ ionophore A23187, 1 mM EGTA, and either no added CaCl 2 (for background) or 0.9 mM CaCl 2 (giving a free Ca 2ϩ concentration of 3 M). Following subtraction of background, the molecular turnover rate was calculated as the ratio between the rate of ATP hydrolysis and the active-site concentration, using as active-site concentration the maximum amount of phosphoenzyme that can be formed from ATP (cf. Fig. 1), and the resulting data are shown relative to wild type (turnover rate 110 s Ϫ1 ).
b Measurement of ATP-driven Ca 2ϩ transport was carried out by filtration following incubation for 5 min at 37°C in a medium containing 20 mM MOPS/Tris (pH 6.8), 100 mM KCl, 5 mM ATP, 5 mM MgCl 2 , 0.5 mM EGTA, 0.45 mM 45 CaCl 2 , and 5 mM potassium oxalate to trap Ca 2ϩ in the lumen of the microsomal vesicles. To correct for expression level, the data were related to the active-site concentration as described above for ATP hydrolysis, and the results are shown relative to wild type.
c Data obtained as for Fig. 1. toward the cytoplasmic side of the membrane. It is believed that the ability to phosphorylate disappears with the dissociation of the first Ca 2ϩ ion in a sequential mechanism (26). Fig.  2 shows such time courses for some of the mutants, and Table  I summarizes the results for all mutants in terms of the halflife of the Ca 2ϩ -bound enzyme form capable of undergoing phosphorylation. The half-lives corresponding to mutants Glu 51 3 Arg/Leu, Glu 55 3 Arg/Leu, Glu 58 3 Arg/Leu, and Asp 59 3 Ala were indistinguishable from that of the wild type, whereas a decrease of around 10-fold was determined for Asp 59 3 Arg/Leu, corresponding to a 10-fold higher rate of Ca 2ϩ dissociation ( Fig. 2 and Table I). Furthermore, the Ca 2ϩ dissociation kinetics was quite sensitive to the size of the hydrophobic side chains in the middle part of M1. Hence, Leu 60 3 Ala/Pro and Val 62 3 Ala displayed markedly reduced half-lives relative to wild type, i.e. corresponding to a 2-to 3-fold increase of the Ca 2ϩ dissociation rate. Most conspicuously, alanine substitution of Leu 65 resulted in an 11-fold increase of the half-life (reduction of the rate of Ca 2ϩ dissociation). Mutation of Phe 57 (to glutamine or alanine) did not affect the Ca 2ϩ dissociation rate to any significant extent ( Fig. 2 and Table I).
Time Course of the E 2 3 [Ca 2 ]E 1 P Transition-To further examine the Ca 2ϩ binding properties of the mutants, the E 2 3 [Ca 2 ]E 1 P transition, consisting of Reactions 1-3 in Scheme 1, was studied by determining the rate of phosphoenzyme formation following simultaneous addition of Ca 2ϩ and [␥-32 P]ATP to enzyme initially present in the Ca 2ϩ -deprived E 2 form at pH 6.0, 25°C. Under these conditions, the E 2 3 [Ca 2 ]E 1 transition is rate-limiting for the phosphorylation, as the reaction with ATP (Reaction 3 in Scheme 1) is much faster (cf. Refs. 22 and 27, also confirmed for the mutants studied here, data not shown). Fig. 3 shows the time course for the wild type and Asp 59 3 Arg, and Table I summarizes the results for all mutants in terms of the rate constants obtained by fitting a monoexponential function to the data. For Asp 59 3 Arg, the observed rate constant was 1.8-fold higher than that corresponding to wild type, suggesting an enhanced rate of the E 2 3 [Ca 2 ]E 1 transition. For the other mutants the data were indistinguishable from wild type.
Vanadate Dependence of Inhibition of Phosphorylation from [␥-32 P]ATP-Vanadate (VO 4 3Ϫ ), acting as an analogue of the phosphoryl group in the transition state during dephosphorylation, binds preferentially to the E 2 form (28,29). Hence, the apparent affinity for vanadate can be used to quantify mutational effects on the equilibrium between E 2 and E 1 conformations, provided that the intrinsic affinity of the E 2 form for vanadate is not affected by the mutations. We have previously devised an assay in which the enzyme is equilibrated with various concentrations of vanadate at 25°C in the absence of  Table I together with the corresponding values for other mutants obtained in the same way. Ca 2ϩ and ATP, followed by determination of the amount of phosphoenzyme formed upon the addition of Ca 2ϩ and [␥-32 P]ATP at 0°C (23,30). Because vanadate binding and phosphorylation are competitive, and vanadate dissociation is very slow at 0°C, the amount of phosphoenzyme formed from ATP under these conditions reflects the proportion of enzyme present in the vanadate-free form before the addition of Ca 2ϩ and ATP. Using this assay, the apparent affinity constants for vanadate were determined for mutants with altered Ca 2ϩ binding characteristics and for the Phe 57 mutants, and are listed in Table II. The titration data are shown in Fig. 4 for wild type and Asp 59 3 Arg, which displayed a strikingly reduced appar-ent vanadate affinity compared with that of the wild type. This is consistent with a displacement of the E 2 -E 1 equilibrium in favor of E 1 and suggests that the above-described enhancement of the E 2 3 [Ca 2 ]E 1 transition rate in Asp 59 3 Arg is caused by an enhanced rate of the E 2 3 E 1 conformational change of Ca 2ϩ -free enzyme (Reaction 1 in Scheme 1). Among the other mutants, Leu 60 3 Ala and Val 62 3 Ala showed a slight (2-fold) increase in vanadate affinity, suggesting that there could be a displacement of the E 2 -E 1 equilibrium in favor of E 2 .
Time Course of Dephosphorylation of Phosphoenzyme Formed from [␥-32 P]ATP-To study the effects of the mutations on the processing of the phosphoenzyme, we first followed the decay of phosphoenzyme formed from [␥-32 P]ATP at pH 7.0, 0°C, presence of K ϩ , i.e. conditions where it is well recognized that the [Ca 2 ]E 1 P 3 Ca 2 E 2 P transition (cf. Reaction 4 in Scheme 1) is the major rate-limiting step during phosphoenzyme turnover of the wild-type enzyme (31,32). To observe the dephosphorylation, phosphorylation from [␥-32 P]ATP was terminated by simultaneous addition of EGTA and excess non-labeled ATP. Fig. 5 shows the phosphoenzyme decay for some of the mutants, and Table II summarizes the results for all mutants in terms of the rate constants obtained by fitting a monoexponential decay function to the data. The rate of disappearance of phosphoenzyme was wild type-like in mutants Glu 51 3 Arg/Leu, Glu 55 3 Arg/Leu, and Glu 58 3 Arg/Leu, as well as Asp 59 3 Arg. In contrast, a reduction of the rate of phosphoenzyme decay to only 17% was seen for Asp 59 3 Leu, whereas alanine substitution of this residue resulted in a much smaller decrease (to 68%). Among the mutants with alteration to hydrophobic residues, the most pronounced reduction of the rate of dephosphorylation (to 9%) was observed for Leu 65 3 Ala, whereas Leu 60 3 Ala and Val 62 3 Ala were slightly affected in the opposite direction (1.7-fold increase). To distinguish between effects on Reactions 4 -6 in Scheme 1, we determined the ADP sensitivity of the phosphoenzyme intermediate accumulated after phosphorylation with [␥-32 P]ATP, i.e. its ability to react with ADP and donate the phosphoryl group back to ADP, forming ATP. Only [Ca 2 ]E 1 P is able to undergo this were performed using Bio-Logic quenched flow module QFM-5 or SFM-400/Q according to previously described mixing protocols (22). Wildtype or mutant enzyme preincubated in 40 mM MES/Tris (pH 6.0), 80 mM KCl, and 2 mM EGTA, was mixed with an equal volume of 40 mM MES/Tris (pH 6.0), 80 mM KCl, 10 mM MgCl 2 , 2.2 mM CaCl 2 , and 10 M [␥-32 P]ATP, followed by acid quenching at the indicted times. A monoexponential function was fitted to the data, and the results were normalized separately for wild type and mutant, taking the phosphorylation corresponding to infinite time as 100%. The (relative) rate constant is shown in Table I together with the corresponding values for other mutants obtained in the same way.  reaction. Following phosphorylation of wild-type and mutant Ca 2ϩ -ATPase under conditions identical to those described for Fig. 5 5 and 6 in Scheme 1). The reduced rate of phosphoenzyme decay observed for these mutants in Fig. 5 suggests the latter possibility. To clarify this issue further, we measured directly the dephosphorylation of the E 2 P form (Figs. 6 and 7), as well as the rate of 3Ϫ ] n / (K 0.5 n ϩ [VO 4 3Ϫ ] n )) was fitted to the data, and the results were normalized separately for wild type and mutant, taking EP max as 100%. The (relative) K 0.5 value is shown in Table II  To follow the dephosphorylation, the phosphoenzyme was chased by addition of 6.7 mM EGTA with 1 mM non-radioactive MgATP followed by acid quenching at the time intervals indicated. A monoexponential decay function was fitted to the data, and the maximal phosphorylation was taken as 100%. The (relative) rate constants are listed in Table II together with the corresponding values for other mutants obtained in the same way.
FIG. 6. Time course of dephosphorylation of E 2 P phosphoenzyme at pH 6.0. Phosphorylation was carried out at 25°C for 10 min in 100 mM MES/Tris (pH 6.0), 10 mM MgCl 2 , 2 mM EGTA, 30% (v/v) Me 2 SO, and 0.5 mM 32 P i . Subsequently, the phosphoenzyme was chased by a 19-fold dilution of an aliquot into a medium (kept at 25°C) containing 100 mM MES/Tris (pH 6.0), 2 mM EGTA, 10 mM EDTA, 15% Me 2 SO, and 0.5 mM non-radioactive P i , and acid quenching was performed at the indicated time intervals. A monoexponential decay function was fitted to the data, and the maximal phosphorylation was taken as 100%. Each mutant is shown together with wild-type data obtained in the same series of experiments, and the (relative) rate constants are listed in Table II Fig. 6, except that the chase medium was kept at 0°C and contained 40 mM MOPS/Tris (pH 7.0), 5 mM MgCl 2 , 2 mM EGTA, 80 mM KCl, and 0.5 mM non-radioactive P i . accumulation of ADP-insensitive phosphoenzyme upon phosphorylation with ATP ( Fig. 8 and Table II).
Phosphoenzyme Formed from 32 P i -Under favorable conditions (absence of Ca 2ϩ , pH 6.0, 25°C, presence of the organic solvent dimethyl sulfoxide, absence of alkali metal ions), it is possible to form the phosphoenzyme intermediate E 2 P by "backward" reaction of E 2 with inorganic phosphate (cf. Reaction 6 in Scheme 1), even if the latter is present only at submillimolar concentration. The P i concentration dependence of the amount of E 2 P formed this way showed characteristics similar to wild type for Glu 51 3 Arg/Leu, Glu 55 3 Arg/Leu, and Glu 58 3 Arg/Leu (Table II). Arginine and leucine substitution of Asp 59 affected K 0.5 for P i in opposite directions, the former causing a 6-fold increase and the latter a decrease relative to wild type. No significant difference was seen between Asp 59 3 Ala and the wild type. A 2-fold increase of K 0.5 for P i was seen with Phe 57 3 Gln/Ala and Val 62 3 Ala, and in Leu 60 3 Pro the K 0.5 was 4-fold increased. Remarkably, the K 0.5 was lowered around 3-fold (apparent P i affinity increased) in Leu 65 3 Ala.
The dephosphorylation kinetics of E 2 P phosphoenzyme (cf. Reaction 6 in Scheme 1), formed under the conditions just described, was studied for some of the mutants by a 19-fold dilution into a chase medium, which terminated the phosphorylation from 32 P i . Fig. 6 shows the time course of E 2 P dephosphorylation at 25°C in medium containing 15% dimethyl sulfoxide at pH 6.0 in the absence of K ϩ , i.e. conditions where this reaction is rather slow and, therefore, easy to follow, and which are rather similar to those under which the K 0.5 values for P i were obtained. The rate constants determined by fitting a monoexponential function are listed in Table II. The variation of the k obs for E 2 P dephosphorylation paralleled to a large extent the change in K 0.5 for P i . Hence, the variation of the K 0.5 for P i is largely due to the mutational effects on E 2 P dephosphorylation. In particular, an 8-fold increase of k obs was determined for Asp 59 3 Arg, whereas Asp 59 3 Leu showed a 4-fold decrease (Fig. 6). Leu 60 3 Pro showed a 5-fold enhanced k obs , whereas Phe 57 3 Gln/Ala, Leu 60 3 Ala, and Val 62 3 Ala showed minor changes relative to wild type (up to 2.3-fold). A most conspicuous, almost complete block of E 2 P dephosphorylation was seen for Leu 65 3 Ala (Fig. 6 and Table II).
The markedly reduced k obs values for E 2 P dephosphorylation of Asp 59 3 Leu and Leu 65 3 Ala seem to explain perfectly well the accumulation of ADP-insensitive phosphoenzyme (E 2 P) observed for these mutants in the phosphorylation experiments with ATP described above. These experiments were, however, performed at pH 7.0, 0°C, and in the presence of K ϩ , whereas the results presented in Fig. 6 were obtained under conditions that deliberately slowed the dephosphorylation of E 2 P. This led us to study the dephosphorylation of E 2 P further for selected mutants at pH 7.0, 0°C, presence of K ϩ , and the results are seen in Fig. 7 and Table II. Clearly, the dephosphorylation of E 2 P was found markedly inhibited in Asp 59 3 Leu and Leu 65 3 Ala also under these conditions (ϳ10-and 33-fold, respectively, relative to wild type).
Time Course of the [Ca 2 ]E 1 P 3 Ca 2 E 2 P Transition-Because the data in Fig. 7 show that E 2 P 3 E 2 is the major rate-limiting step in the dephosphorylation of mutants Asp 59 3 Leu and Leu 65 3 Ala, even at pH 7.0, 0°C, presence of K ϩ , the results in Fig. 5 do not provide information about the rate of the [Ca 2 ]E 1 P 3 Ca 2 E 2 P transition (cf. Reaction 4, Scheme 1) in these mutants. Such information was instead obtained using an experimental design that takes advantage of the ADP-sensitivity of [Ca 2 ]E 1 P. The enzyme was phosphorylated with [␥-32 P]ATP under conditions known to slow down dephosphorylation of E 2 P, such that E 2 P accumulates even in the wild type (0°C, pH 8.0, presence of Li ϩ instead of K ϩ , high Mg 2ϩ / Ca 2ϩ ratio). After various phosphorylation intervals, the amount of ADP-insensitive phosphoenzyme was determined as the phosphoenzyme remaining after a 4-s incubation with ADP to remove [Ca 2 ]E 1 P. Because the [Ca 2 ]E 1 3 [Ca 2 ]E 1 P reaction is very rapid, the rate of appearance of ADP-insensitive phosphoenzyme corresponds to that of the [Ca 2 ]E 1 P 3 Ca 2 E 2 P transition under these conditions. Interestingly, the rates determined for Asp 59 3 Leu and Leu 65 3 Ala in this way were, respectively, 3.4-and 4.2-fold higher than that of the wild type ( Fig. 8 and Table II). This constitutes a second factor, besides the block of E 2 P dephosphorylation, contributing to the high steady-state concentration of ADP-insensitive phosphoenzyme in these mutants. DISCUSSION The present results provide the first functional evidence that transmembrane segment M1 of the Ca 2ϩ -ATPase is critical to Ca 2ϩ interaction and to phosphoenzyme turnover. During active Ca 2ϩ transport, the Ca 2ϩ -ATPase binds Ca 2ϩ from the cytoplasmic side of the membrane, transfers the ions across the membrane, and releases them on the luminal side. It is generally believed that the translocation of Ca 2ϩ across the membrane involves an "occluded" state, where the ions are inaccessible to the medium on either side of the membrane and their dissociation restricted by structural components acting as gates. In the occluded state, Ca 2ϩ is bound by residues in M4, M5, M6, and M8 (8, 10 -12). The Ca 2ϩ ions may become occluded within the protein concomitantly with phosphorylation from ATP (5,33). Evidence has, however, been presented that the Ca 2ϩ ions are not only occluded in the phosphoenzyme, but also most of the time in the non-phosphorylated Ca 2ϩ -bound form (6), as indicated by the brackets in Scheme 1. This seems to agree with the crystal structure of the Ca 2ϩ -ATPase with bound Ca 2ϩ , revealing no obvious Ca 2ϩ entry or exit pathways (8). Furthermore, the fact that Ca 2ϩ can dissociate only very slowly from the enzyme complex with CrATP, even though the ␥-phosphoryl group is not transferred to the enzyme in this complex, indicates that phosphorylation is not required for occlusion of Ca 2ϩ (7). The present findings are consistent with the existence of a Ca 2ϩ -occluded non-phosphorylated state, whose stability depends on the structural properties of M1. Our measurements indicate a 10-fold increase of the rate of Ca 2ϩ mM EGTA, and 2 mM ADP was added to remove ADP-sensitive phosphoenzyme, followed by acid quenching 4 s later. A monoexponential function was fitted to the data, and the results were normalized separately for wild type and mutant, taking the phosphorylation corresponding to infinite time as 100%. The (relative) rate constant is shown in Table II together with the corresponding value for Asp 59 3 Leu obtained in the same series of experiments. dissociation from this state toward the cytoplasmic side in mutants Asp 59 3 Arg and Asp 59 3 Leu, whereas Ca 2ϩ dissociation was wild type-like in the mutants with arginine or leucine substitution of Glu 51 , Glu 55 , or Glu 58 (Fig. 2 and Table  I). The Phe 57 mutants also displayed wild type-like Ca 2ϩ dissociation, whereas Leu 60 3 Ala/Pro and Val 62 3 Ala caused an acceleration of Ca 2ϩ dissociation, although to a lesser extent than the Asp 59 mutations. Moreover, a remarkable 11-fold reduction of the rate of Ca 2ϩ dissociation toward the cytoplasmic side was observed for Leu 65 3 Ala. This demonstration of the role of M1 in Ca 2ϩ occlusion was made feasible by the quenched flow technique that allows measurement of Ca 2ϩ dissociation from the [Ca 2 ]E 1 form toward the cytoplasmic side, even with the relatively small amounts of enzyme that can be harvested from the cell culture. Thus, the rate of Ca 2ϩ dissociation from the [Ca 2 ]E 1 form can be both increased and decreased by mutation within the middle part of M1 (C-terminal to the bend of M1 seen in the E 2 -Tg crystal structure), and our results suggest that the middle part of M1, but not the most N-terminal part containing Glu 51 , Glu 55 , and Glu 58 , is important in control of the gates at the Ca 2ϩ occlusion sites.
As regards the proposed role of Glu 51 , Glu 55 , and Glu 58 in the Ca 2ϩ entry pathway (14), the results reported here clearly argue against any involvement of these glutamate side chains in Ca 2ϩ recognition and binding, and it is not likely that they contribute to a Ca 2ϩ selectivity filter. Furthermore, the finding that substitution of Glu 58 by arginine left the Ca 2ϩ binding properties of the Ca 2ϩ -ATPase unaffected questions the existence of a close interaction between Glu 58 in M1 and Glu 309 at Ca 2ϩ binding site II, such as seen in the published crystal structure of the Ca 2ϩ -bound enzyme (8). A possible reason for this apparent discrepancy could be a high degree of thermal mobility of the Glu 58 side chain. It is also possible that the [Ca 2 ]E 1 form adopted in the native state differs more profoundly from the crystal structure.
The reduced apparent affinity of mutant Asp 59 3 Arg for Ca 2ϩ activation of phosphorylation ( Fig. 1) reflects the enhanced rate of Ca 2ϩ dissociation from [Ca 2 ]E 1 and not a displacement of the E 2 -E 1 equilibrium in favor of the low affinity E 2 form. In fact, the data in Figs. 3 and 4 suggest that mutation Asp 59 3 Arg enhances the rate of the E 2 3 E 1 conformational change, leading to accumulation of E 1 with resulting low sensitivity to vanadate inhibition of phosphoenzyme formation. Because release of counter-transported protons from E 2 may be rate-limiting for the E 2 3 E 1 transition (27), the enhancement of this transition suggests that proton dissociation from the transport sites, like Ca 2ϩ dissociation, is facilitated by the Asp 59 3 Arg mutation.
For mutants Leu 60 3 Ala/Pro and Val 62 3 Ala, the enhanced rate of Ca 2ϩ dissociation likewise seems to result in a reduced apparent affinity for Ca 2ϩ activation of phosphorylation. In addition, there could be a contribution to the reduced apparent affinity for Ca 2ϩ from displacement of the E 2 -E 1 equilibrium in favor of the low affinity E 2 form in Leu 60 3 Ala and Val 62 3 Ala, because the vanadate affinity was slightly increased in these mutants (Table II). The apparent affinity for Ca 2ϩ in activation of phosphorylation was normal in Asp 59 3 Leu, despite the increased Ca 2ϩ dissociation rate (Figs. 1 and 2). This may be accounted for by a considerably reduced rate of dephosphorylation (Figs. 5-7). As previously reported, the effect of a low rate of phosphoenzyme turnover on the apparent affinity for Ca 2ϩ activation of phosphorylation can be understood on the basis of computer simulations of the Ca 2ϩ -ATPase reaction cycle (34). According to the computational analysis, an increased apparent affinity for Ca 2ϩ is actually expected when the rate of phosphoenzyme turnover is reduced, because a lower phosphorylation rate (i.e. Ca 2ϩ saturation) is required to maintain a certain level of phosphorylation under these conditions. Hence, for Asp 59 3 Leu, the increased rate of Ca 2ϩ dissociation and the decreased rate of phosphoenzyme turnover act in opposite directions, thereby masking the effects on the apparent Ca 2ϩ affinity.
The importance of the middle part of M1 in the gating at the Ca 2ϩ occlusion sites may be related to the presence of a wateraccessible channel leading between M1 and M3 in the crystal structure of Ca 2ϩ -ATPase in the Ca 2ϩ -free E 2 -Tg form. This channel has the potential of a Ca 2ϩ entry port and is apparently opened by the bending and partial unfolding of the helical structure of M1 at Asp 59 (9). A similar channel may exist in the native non-crystalline protein and function as a migration pathway for Ca 2ϩ in one or more of the conformations that precede the occluded [Ca 2 ]E 1 form in the process of Ca 2ϩ binding (E 2 , E 1 , and CaE 1 ). Because the channel leads to Glu 309 at Ca 2ϩ site II, it may provide a passage for the Ca 2ϩ ion that binds at site II (i.e. the one that binds last in the sequential mechanism). In line with this assignment, the presently observed mutational effects on the rate of disappearance of ability to phosphorylate from ATP upon addition of EGTA (Fig. 2) reflect changes of the rate of dissociation of the Ca 2ϩ ion that leaves first in the sequential mechanism, i.e. the one that was bound last (26,35). The Ca 2ϩ ion that binds first (at site I, cf. Ref. 12) might enter (and leave upon dissociation) by a different route. Some evidence has actually been presented suggesting the existence of a pre-binding site for Ca 2ϩ in the loop between transmembrane segments M6 and M7 ("L6 -7"), which could be related to the entry port for the Ca 2ϩ ion binding at site I (36).
Comparison of the two crystal structures of the Ca 2ϩ -ATPase indicates that the transition from [Ca 2 ]E 1 to E 2 -Tg is accompanied by a lateral and upward (toward the cytoplasmic side) movement of M1 in the membrane, and bending of the helix at Asp 59 , which probably is caused by steric collision with M3 (9). In E 2 -Tg, the N-terminal part of M1 containing Glu 51 , Glu 55 , and Glu 58 is bent away from M3, and their side chains define the hydrophilic surface of the amphipathic section. These residues are therefore peripherally located with respect to the proposed Ca 2ϩ entrance. A similar position of this part of M1 in the native non-crystalline protein could explain the lack of importance of the glutamate side chains for Ca 2ϩ binding. Asp 59 , on the other hand, is located right at the bending point of M1. Thus, the dramatic effect on Ca 2ϩ dissociation of substitution of Asp 59 by leucine or arginine may result from direct interference with Ca 2ϩ interaction or interference with the movement of M1 that occludes Ca 2ϩ , a movement that conceivably is facilitated by flexibility at Asp 59 . The finding that alanine substitution of Asp 59 is tolerated stresses the requirement for a small side chain that allows movement. The negative charge of the Asp 59 side chain, which might have been expected to participate in directing Ca 2ϩ to the binding sites, seems not to be required for normal Ca 2ϩ interaction.
Among the hydrophobic side chains substituted in the present study, Phe 57 seems less important for the Ca 2ϩ -binding properties and the E 2 3 E 1 conformational transition than expected if it were crucial to the attachment of the bent part of M1 to the lipid bilayer and, thereby, to the positioning of this part of M1. On the other hand, the lack of a significant effect on Ca 2ϩ binding of substitution of Phe 57 may be considered consistent with its location away from the proposed Ca 2ϩ inlet, on the hydrophobic surface of the bent part of M1, and it is conceivable that the removal of a single anchoring side chain is not enough to disrupt the attachment to the lipid bilayer. As regards Leu 60 , Val 62 , and Leu 65 , which are embedded within the membrane, their bulky hydrophobic side chains appear to form an integral part of the wall lining the water-accessible channel seen in the E 2 -Tg structure, with Leu 65 at the bottom. Hence, our finding of significant changes of the Ca 2ϩ dissociation rate upon substitution of these residues with the smaller alanine seems to be compatible with the hypothesis that this channel serves as a migration pathway for Ca 2ϩ . The effects of hydrophobic size reduction in this part of M1 could simply be due to local changes in the proportions of the channel, providing a more free passage for the dissociating Ca 2ϩ ions in case of Leu 60 3 Ala/Pro and Val 62 3 Ala and collapsing the channel in case of the Leu 65 3 Ala mutation. Of course, it cannot be excluded that changes to M1 exert more distant effects on a Ca 2ϩ migration pathway located elsewhere in the protein.
In the crystal structure of the Ca 2ϩ -bound enzyme, Leu 65 is below the level of the Ca 2ϩ ions (i.e. closer to the luminal surface), and it seems that its bulky side chain may contribute to form a luminal gate, preventing the immediate transport of the ions to the lumen upon binding from the cytoplasm. A premature transfer of the Ca 2ϩ ions to the lumen caused by a defective luminal gate in Leu 65 3 Ala (i.e. rapid formation of an enzyme form somewhat similar to Ca 2 E 2 P) could account for the rapid disappearance of ADP sensitivity of the phosphoenzyme in this mutant (Fig. 8), because the ADP sensitivity is thought to be specifically associated with the [Ca 2 ]E 1 P form. Interestingly, also Asp 59 3 Leu showed an enhanced rate of disappearance of ADP sensitivity of the phosphoenzyme (Table  II), which again might indicate premature Ca 2ϩ transfer to luminally facing sites. In addition to the enhanced rate of disappearance of ADP sensitivity of the phosphoenzyme, Leu 65 3 Ala and Asp 59 3 Leu both showed a conspicuous block of the dephosphorylation of E 2 P, the rate being reduced 33-fold and 4-to 10-fold (depending on pH), respectively (Table II). Hence, there is a preference for forms with luminally facing Ca 2ϩ sites and ADP insensitivity of the phosphoenzyme (Ca 2 E 2 P and E 2 P) in both of these mutants. A difference between these mutants is, however, that Ca 2ϩ dissociation toward the cytoplasmic side is increased in Asp 59 3 Leu, whereas it is inhibited in Leu 65 3 Ala (Fig. 2).
Some of the other mutations also affected the processing of the phosphoenzyme (Reactions 4 -6 in Scheme 1). Hence, Leu 60 3 Ala and Val 62 3 Ala showed a slightly enhanced rate of dephosphorylation of phosphoenzyme formed from ATP ( Fig. 5 and Table II), probably reflecting enhancement of the rate-limiting [Ca 2 ]E 1 P 3 Ca 2 E 2 P transition. The rate of E 2 P dephosphorylation was markedly increased in Asp 59 3 Arg (8-fold) and Leu 60 3 Pro (4-to 5-fold) and to a lesser extent in Phe 57 3 Ala/Gln and Val 62 3 Ala (about 2-fold, Table II). It is noteworthy that the replacement of Asp 59 with arginine and leucine led to opposite effects on E 2 P dephosphorylation, which may be attributable to the difference in charge/polarity, resulting in different potentials for salt bridge formation or interaction with the lipid bilayer. The leucine may fix this part of M1 in close contact with the lipid bilayer, whereas the arginine may tend to draw it into the solvent phase. Taken together, the effects of M1 mutations on phosphoenzyme processing demonstrate that M1, besides being important for Ca 2ϩ interaction, furthermore is a critical element in the long range signaling between the transmembrane domain and the catalytic site in the cytoplasmic part of the Ca 2ϩ -ATPase molecule. Several pieces of evidence suggest that the A-domain undergoes large movements during the catalytic cycle, being separated from the P-domain in [Ca 2 ]E 1 and [Ca 2 ]E 1 P and in rather close contact with this domain in E 2 and E 2 P, probably contributing to the catalytic site in these forms (9,(37)(38)(39). Because M1 is physically connected to the A-domain through a flexible hinge segment at the N-terminal end and through M2 at the other end, it is tempting to speculate that the movement of the A-domain is closely coupled with movement of M1, and that the effects of the M1 mutations on the processing of the phosphoenzyme, described here, are brought about by an altered position and/or reduced motional freedom of the A-domain, caused by changes of the position and structure of M1.
In conclusion, our results provide evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of Ca 2ϩ from its membranous binding sites. Our results further indicate that these residues are also critical to the proper processing of the phosphoenzyme and, thus, to the long range transmission of conformational changes to the cytoplasmic catalytic site.


extfieldsofstudy: Biology Medicine

text:
Xenoestrogen-induced ERK-1 and ERK-2 activation via multiple membrane-initiated signaling pathways.

Xenoestrogens can mimic or antagonize the activity of physiological estrogens, and the suggested mechanism of xenoestrogen action involves binding to estrogen receptors (ERs). However, the failure of various in vitro or in vivo assays to show strong genomic activity of xenoestrogens compared with estradiol (E2) makes it difficult to explain their ability to cause abnormalities in animal (and perhaps human) reproductive functions via this pathway of steroid action. E2 has also been shown to initiate rapid intracellular signaling, such as changes in levels of intracellular calcium, cAMP, and nitric oxide, and activations of a variety of kinases, via action at the membrane. In this study, we demonstrate that several xenoestrogens can rapidly activate extracellular-regulated kinases (ERKs) in the pituitary tumor cell line GH3/B6/F10, which expresses high levels of the membrane receptor for ER-alpha (mER). We tested a phytoestrogen (coumestrol), organochlorine pesticides or their metabolites (endosulfan, dieldrin, and DDE), and detergent by-products of plastics manufacturing (p-nonylphenol and bisphenol A). These xenoestrogens (except bisphenolA) produced rapid (3-30 min after application), concentration (10(-14)-10(-8) M)-dependent ERK-1/2 phosphorylation but with distinctly different activation patterns. To identify signaling pathways involved in ERK activation, we used specific inhibitors of ERs, epidermal growth factor receptors, Ca2+ signaling, Src and phosphoinositide-3 kinases, and a membrane structure disruption agent. Multiple inhibitors blocked ERK activation, suggesting simultaneous use of multiple pathways and complex signaling web interactions. However, inhibitors differentially affected each xenoestrogen response examined. These actions may help to explain the distinct abilities of xenoestrogens to disrupt reproductive functions at low concentrations.

Xenoestrogens are a large and structurally diverse group of compounds, which can act as inappropriate estrogens and/or can interfere with the actions of endogenous estrogens such as estradiol (E 2 ) or other reproductive steroids. Many studies have demonstrated that contamination of the environment or treatment with xenoestrogens can disrupt developmental programs to alter sexual phenotypes and reproductive functions. Examples of such disruptions are a decline in the sperm quality of fish, interference with the sexual development of alligators and turtles, disruption of pregnancies in laboratory animals, interference with blastocyst implantation, and inappropriately induced progesterone receptor expression and uterine weight increases [reviewed in Witorsch (2002)]. In addition, environmental estrogens have been shown to inhibit the human sperm acrosomal reaction (Turner et al. 1997), and xenoestrogens are also suspected of causing breast cancer cell and vaginal epithelial cell proliferation (Krishnan et al. 1993;Long et al. 2000).
Most previous studies investigated the possible actions of xenoestrogens through classical nuclear estrogen receptors (ERs) modifying gene activity (Long et al. 2000;Massaad and Barouki 1999;McLachlan 1993;Rosselli et al. 2000;Sonnenschein et al. 1995). A variety of in vivo and in vitro screening assays have been applied to predict the estrogenic potential of xenoestrogens, including several receptor-reporter-gene assay systems in transfected mammalian or yeast cell lines (Bonefeld-Jorgensen et al. 2001;Elsby et al. 2001;Gutendorf and Westendorf 2001;Hodges et al. 2000;Inoue et al. 2002;Lee et al. 2002;Massaad and Barouki 1999;Rajapakse et al. 2002;Willard and Frawley 1998). However, these methods demonstrated that xenoestrogens act very weakly (only at concentrations 1,000-to 10,000-fold higher than E 2 ), if at all, via genomic activation pathways. Other tests have been assumed to assess genomic responses, but may in fact be assays for either nongenomic or combination mechanisms. These include cell proliferation test systems (Gutendorf and Westendorf 2001;Hodges et al. 2000;Rousseau et al. 2002;Soto et al. 1994), receptor binding assays for ER-α or ER-β (Granek and Rishpon 2002;Gutendorf and Westendorf 2001;Lee et al. 2002), and predictions of ligand binding affinity and conformation of ER complexes with xenoestrogens by computer modeling (Suzuki et al. 2001;van Lipzig et al. 2004;Yu et al. 2002).
In addition to the classical genomic pathway, steroids can produce rapid (within a few minutes after application) nongenomic signaling effects via second messenger systems, for example, Ca 2+ , K + , cAMP, and nitric oxide level changes; activation of G protein-mediated events; and stimulation of different types of kinases such as extracellular-regulated kinases (ERKs), phosphoinositide-3 kinase (PI3K), p38, and Jun kinase (Junk) (Aronica et al. 1994;Doolan and Harvey 2003;English et al. 1999;Filardo et al. 2000;Haynes et al. 2003;Kelly et al. 1999;Nadal et al. 1998;Prevot et al. 1999;Razandi et al. 2003). Although the precise molecular mechanisms of nongenomic actions are not fully understood, it is known that some rapid E 2 effects can be initiated by ligand binding to membrane-associated ERs (mERs) that have been shown to be the same proteins as their nuclear receptor counterparts in several systems (Chen et al. 1999;Levin 1999;Norfleet et al. 1999;Pappas et al. 1994). Therefore, it is reasonable to suggest that xenoestrogens can bind to mER and produce rapid changes in signaling, similar to E 2 . However, there are few data (Adeoya-Osiguwa et al. 2003;Nadal et al. 2000;Ruehlmann et al. 1998;Sato et al. 2003;Watson et al. 1999a;Wober et al. 2002) addressing the ability of environmental estrogens to mediate nongenomic estrogenic actions, and many studies on this phenomenon have used concentrations of xenoestrogens much higher than would be found in contamination sites.
In the present study, we investigated the ability of some of these estrogen mimetics (belonging to the major classes of environmental estrogens) to produce rapid activation of ERKs via various signaling pathways in the GH 3 /B6/F10 prolactinoma cell line. We previously selected this subline for its robust expression of mER-α and its capacity for rapid E 2 -induced prolactin release (Pappas et al. 1994), and we also demonstrated that adequate levels of mER were necessary to elicit rapid ERK responses (Bulayeva et al. 2004). Here, we tested organochlorine pesticides Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004 [dieldrin, endosulfan, and the DDT metabolite o,p´-dichlorodiphenylethylene (DDE)], detergents used in plastics manufacturing (p-nonylphenol and bisphenol A), and the natural phytoestrogen coumestrol. The signaling pathways that we examined are generally known to initiate at the plasma membrane and go through multiple steps before converging on the ERKs. Others have associated features such as G protein involvement, Ca 2+ influx, and epidermal growth factor receptor (EGFR) phosphorylation with downstream ERK activations, which can lead to diverse cellular functions such as cell proliferation, transformation, differentiation, and migration (Belcheva and Coscia 2002;Razandi et al. 2003). Xenoestrogens, such as endosulfan and chlordane  and nonylphenol, bisphenol A, and coumestol (Gutendorf and Westendorf 2001), at relatively low (10 -9 -10 -7 M) concentrations, can produce proliferation in other cell lines, and this function can be regulated by the xenoestrogen benzopyrene through ERK (Tsai et al. 2004), The alteration of cell proliferation, as well as other functions downstream of ERK activation, could lead to endocrine disruptions known to be caused by environmental estrogens.
To monitor signaling events leading to ERK activation, we used our fixed cell-based ELISA (Bulayeva et al. 2004), which allows us to analyze many samples and thus detailed time-and concentration-dependent changes in ERK phosphorylation resulting from actions of multiple xenoestrogenic compounds and specific inhibitors of signaling cascade participants. Such comparative explorations of differential signaling pathway involvement, kinetics, and potencies unique to each compound may lead to more detailed predictions about the hazards of exposure mediated through different subsets of responses related to endocrine disruption by individual xenoestrogens.
Cell culture. Our clonal rat prolactinoma cell line GH 3 /B6/F10 was selected for high expression of mER-α (Pappas et al. 1994). Cells were routinely subcultured in DMEM containing 12.5% horse serum, 2.5% defined supplemented calf serum, and 1.5% fetal calf serum. For individual experiments, cells were deprived of steroids for 48 hr after plating by substituting DMEM containing 1% charcoalstripped (4×) serum. All test estrogens were dissolved in ehanol (EtOH) at a 10 -2 M concentration to create a stock solution and then diluted into experimental media to yield final concentrations from 10 -8 to 10 -12 M. The EtOH concentration used as the vehicle control was 0.0001%.

Fixed cell-based ELISA.
To estimate ERK phosphorylation quantitatively, we used a cellbased ELISA, which we previously developed and described (Bulayeva et al. 2004). Briefly, cells (10 4 cells/well) were plated in 96-well plates (Corning Incorporated, Corning, NY) and withdrawn from serum hormones by incubation in medium containing 1% charcoalstripped serum for 48 hr before experiments began. The cells were next treated with hormones and estrogen mimetics for 3-30 min, and then fixed with 2% paraformaldehyde/ 0.2% picric acid at 4°C for 48 hr. After fixation, the cells were incubated with phosphatebuffered saline (PBS) containing 2% bovine serum albumin (BSA) and 0.1% Triton X-100 for 1 hr at room temperature (RT), and then with primary Ab against pERK (1:400 in PBS/1% BSA/0.1% Triton X-100) overnight at 4°C. After a wash with PBS, biotin-conjugated secondary Ab (1:300) in PBS/1% BSA was added for 1 hr at RT. The cells were again washed in PBS and incubated with Vectastain ABC-AP solution (100 µL/well) for 1 hr at RT, and then Vectastain alkaline phosphatase substrate (pNpp solution) with levamisole was added to each well (100 µL). Plates were incubated in the dark for 30 min at 37°C, and the signal from para-nitrophenol (pNp) was read at A 405 in a model 1420 Wallac microplate reader (Perkin Elmer, Boston, MA). The pNp signal was normalized to cell number, determined by using the crystal violet (CV) assay (Campbell and Watson 2001). Briefly, after washing away alkaline phosphatase reaction reagents with double-distilled H 2 O, the plate was completely dried at RT. CV solution (0.1% in water, filtered) was added at 50 µL/well, incubated for 30 min at RT, and washed out with double-distilled H 2 O. Dye was released from the cells with 50 µL/well acetic acid (10% in water) at RT for 30 min. The A 590 signal was then read in the microplate reader.
Statistics. Data were compared for significance of differences using Sigma Stat 3 (Jandel Scientific, San Rafael, CA) and oneway analysis of variance (significance accepted at p ≤ 0.05).

Results
Xenoestrogens can cause unique time-dependent patterns of ERK phosphorylation. E 2 (10 -9 M) produced rapid (3, 15, and 30 min after application) and bimodal (with apparent periods of dephosphorylation between activation periods) ERK phosphorylation. Xenoestrogens at 10 -9 M also caused ERK activations but with distinct temporal patterns (Figure 1). According to these patterns, compounds could be divided into several groups. Endosulfan and nonylphenol did not cause an initial (3 min) stimulation, but instead caused only a delayed single ERK phosphorylation peak at 30 min (which we designated slow-phase-only responders). DDE and dieldrin caused a single peak of activation at 6-10 min and were unable to cause a second sustained activation at 30 min (fastphase-only responders). Coumestrol produced a rapid response (significant by 6 min), but the phosphorylation levels never declined after the activation, as was seen with the other active compounds. Bisphenol A did not produce any significant changes from the basal level of ERK phosphorylation during the 30 min assessment time and was not examined further in this study. All active xenoestrogens produced only a monophasic activation, failing to mimic the bimodal E 2 activation.
Xenoestrogens can be potent activators of ERK phosphorylation but with unique concentration-dependent patterns. At optimal stimulation time points (Figure 1), different concentrations of E 2 and xenoestrogens were compared in their ability to activate ERKs ( Figure 2). E 2 (tested at 3 min) was active in two concentration ranges: very low levels (10 -14 M) and higher, but still physiological, levels (10 -9 -10 -8 M). Nonylphenol and coumestrol showed similar patterns of potency, with dual ranges of activation similar to that seen with E 2 . Endosulfan was able to produce phosphorylation at almost all tested concentrations but still showed an apparent loss of activity centered on the 10 -10 M concentration. DDE and dieldrin were not active at low concentrations (picomolar and lower) but were active in the concentration range centering on 10 -9 M. Thus, although some subtle differences were observable between activation patterns for each compound, basically two patterns of stimulation were seen: compounds active in both the subpicomolar and nanomolar ranges (E 2 , endosulfan, nonylphenol,

Article | Xenoestrogen-induced ERK activation
Environmental Health Perspectives • VOLUME 112 | and coumestrol) versus compounds active only in the nanomolar range (DDE and dieldrin).
Possible pathways for ERK activation for different compounds. To detect possible signaling pathways through which E 2 and xenoestrogens could affect pituitary tumor cells, we used inhibitors that have been described in the literature to pinpoint various mechanisms leading to ERK phosphorylation (Belcheva and Coscia 2002;Lowes et al. 2002). ICI and AG 14 are specific antagonists of estrogen and EGFRs, respectively. Nystatin (Nys) is a cholesterol-binding antibiotic that disrupts membrane architecture (Ushio-Fukai et al. 2001). B-TA is a Ca 2+ chelator. PP2 is a Src kinase inhibitor, and Ly is a PI3K inhibitor. An example of each type of xenoestrogen based on temporal activation patterns shown in Figure 1 (fast-phase activator DDE, slow-phase activator endosulfan, and sustained-activator coumestrol) was examined for each of these inhibitor actions. All time points in their activation profiles were examined to determine when each mechanism might come into play (Figures 3-5). Inhibitor data were divided into two groups for clarity of presentation. Figures 3A, 4A, and 5A group together the responses to inhibitor compounds that can interfere with receptors (ERs, EGFRs) or disrupt membrane structures housing receptors: ICI, AG 14, and Nys (group A). Figures 3B,  4B, and 5B group together data for compounds whose substrates are mostly localized in the cell's cytoplasm or are adjacent to the cell membrane and part of the downstream signaling cascades initiated at the membrane: B-TA, PP2, and Ly (group B).
Inhibition of endosulfan-stimulated ERK activation is shown in Figure 3. In these assays, endosulfan stimulated ERK significantly only at 30 min (as in Figure 1). Only ICI and Ly inhibited the endosulfan-provoked ERK activation at 30 min. The activity of ICI implicates ER-α in this process [because this subline does not express ER-β (Campbell and Watson 2001;Norfleet et al. 1999)]. However, even at times when endosulfan could not significantly elevate basal phosphorylation of ERK (3-15 min), all tested inhibitors were able to further deactivate basal ERK activity levels at some of these time points (e.g., AG 14 at 15 min; all group B compounds were effective at 3 and 15 min: PP2 at 6 and 10 min; Ly, ICI, and Nys at 6 min). Such inhibitions are xenoestrogen dependent because the inhibitors alone do not cause these dephosphorylations (Bulayeva et al. 2004).
DDE produced ERK activation only at 6 min (Figure 4), as expected from earlier studies (Figure 1). At this time point, ERK phosphorylation was inhibited by AG 14, PP2, and Ly. Although at other time points DDE did not raise ERK activation levels above basal, the addition of inhibitors nevertheless did lower activity to subbasal levels (all at 3 min; AG 14, PP2, and Ly at 15 min). Altogether, all tested compounds had an effect on basal ERK activity levels at some time point, but some tended to affect this outcome earlier in this time frame compared with others.
Coumestrol activated ERKs from 6 min onward in our assay (as shown in Figure 1F and in Figure 5). During the preactivation phase (3 min), basal levels of phosphorylation were further lowered by ICI, Nys, and B-TA. During the 6 min onward coumestrol activation phase, ICI was never effective at lowering ERK phosphorylation levels. AG 14 was effective at 6-15 min time points, and PP2 during the entire stimulation phase, which suggests early involvement of EGFR and Src kinase. Nys disruption of membrane structure (15-30 min) and Ly inhibition of PI3K (15 min) were effective only during these short temporal windows. B-TA's chelation of Ca 2+ was effective only very late in this sequence, at 30 min. Therefore, most inhibitors were effective at some point, although some later than others.

Discussion
An important and surprising conclusion from our studies was that all tested estrogenic compounds, except bisphenol A, elicited rapid membrane-initiated actions at very low concentrations compared with their reported potencies in classical genomic pathways (Gutendorf and Westendorf 2001;Hodges et al. 2000;Inoue et al. 2002). All active compounds were able to produce rapid (3-30 min) ERK phosphorylations in the nanomolar concentration range, and some (E 2 , coumestrol, nonylphenol, and endosulfan) were also active in the subpicomolar range. Compounds from different classes of endocrine disruptors with dissimilar chemical structures (e.g., endosulfan Article | Bulayeva   as an organochlorine compound vs. nonylphenol as a simple phenolic detergent) can produce the same time-dependent activation pattern for ERKs. Coumestrol, a phytoestrogen, initiated a sustained ERK activation that had no temporal pattern similarity with any of the other tested compounds, including E 2 . None of the tested compounds was able to precisely repeat the E 2 pattern of activation, which may contribute to their disruptive effects on estrogen-mediated endocrine functions.
The bimodal E 2 time-dependent response seems to superimpose the patterns from both groups of other response-producing compounds: fast phase (during the first 10 min) and slow phase (not until 30 min). Interestingly, the most potent endocrine-disrupting chemical in genomic action assays, bisphenol A (Cappelletti et al. 2003;Recchia et al. 2004;Sato et al. 2003), was unable to produce timedependent ERK activation. However, studies in progress show that bisphenol A, although unable to trigger ERK activation, nevertheless is somewhat effective at triggering Ca 2+ influx, resulting in prolactin secretion (Wozniak et al., unpublished data). Thus, there are likely to be specific pathways within the nongenomic signaling network that individual compounds will trigger, leading to different functional end points. Therefore, each xenoestrogenic compound must be tested for an array of possible mechanistic routes of action.
Several tested xenoestrogenic compounds (coumestrol, nonylphenol, and endosulfan) demonstrated a bimodal dose-response curve for ERK activation similar to that seen with E 2 . This is reminiscent of the same bimodal dose-response pattern reported previously for rapid prolactin release after E 2 (Watson et al. 1999b) and E 2 -BSA (Watson et al. 1995) treatment. The reason for this gap in dose responsiveness at intermediate concentrations is still not understood, but it is interesting that other estrogens in the present study demonstrate the same phenomenon. These very low effective doses for xenoestrogens demonstrate that many environmental contamination levels previously thought to be subtoxic may very well exert significant signaland endocrine-disruptive effects, discernable only when the appropriate mechanism is assayed. Possible reasons for these potent effects not being noted previously are that little testing of the nongenomic pathway has been done, many tests did not examine such low concentrations, and some test conditions probably did not adequately remove endogenous estrogen levels (as we have done by use of low quantities of extensively charcoalstripped serum) to reveal effects of these low concentrations. The potent effects we see on nongenomic signaling mechanisms could explain why concentrations previously determined to be inactive via genomic mechanisms still have toxic and teratogenic effects on wildlife (Brucker-Davis et al. 2001). Therefore, the threat levels of these compounds to wildlife, and probably humans, need to be reconsidered.
The complexity of multiple signaling pathways triggered simultaneously is probably related to the organization of ERs within membrane substructures (caveolae or membrane rafts), where they encounter many signaling machineries (Chambliss et al. 2000;Nadal et al. 2000;Razandi et al. 2002). Our data indicate that the disruption of a nongenomic signaling cascade midway in its time course caused by Nys (e.g., for coumestrol) probably corresponds to disruption of this cholesterol-rich meeting place for ligands and receptors with their downstream signaling partners. Interestingly, only endosulfan effects failed to be inhibited by disruption of cholesterol-rich membrane structure, perhaps implicating different membrane subdomains as locations for the actions of different compounds. Alternatively, endosulfan signaling may move into the intracellular compartment rapidly after initiation and earlier than 3 min (and earlier time point assessment using these methods would be technically difficult).
Although here we have only directly assessed ERK activation as a signaling cascade end point, the participation of upstream signaling repertoires was implicated by our specific inhibitor assays. We found that all examined pathways can participate in ERK activation but that different xenoestrogens use different subsets of these pathways. Table 1 summarizes the vulnerability of E 2 - (Bulayeva et al. 2004), endosulfan-, DDE-and coumestrol-initiated actions to inhibitors of different signaling components. E 2 -or xenoestrogentreated cells showed inhibitions of both stimulated ERK phosphorylation levels and background levels of phosphorylation. Our time course measurements allowed an analysis of when pathway inhibitions affected the outcome of ERK phosphorylation, and we noted whether this was very early after treatment (3 min) or later (≥ 6 min). Although these times are arbitrary cutoffs, they allowed us to highlight some possible temporal differences in the effects of compounds' pathways. All xenoestrogens shared activation via all pathways, although compounds differed in their timing of pathway engagement. For example, inhibitors of action via the EGFR and ER were sometimes effective only after 6 min. This could mean that the activation Article | Xenoestrogen-induced ERK activation Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004   Bulayeva et al. (2004). All time points where the combination of xenoestrogen and inhibitor showed a significant reduction in ERK phosphorylation levels below the hormone-untreated background level are shown. sequence took some time to reach the level of a receptor (EGFR is downstream) or that a unique conformation of receptors in the plasma membrane could initially prevent binding by antagonists (ER). Although all xenoestrogens shared activation via the PI3K pathway, PI3K inhibitors could not lower DDE-or coumesterol-mediated ERK phosphorylation levels until ≥ 6 min, so perhaps progression to this level of signaling took variable amounts of time depending upon the compound initiating the response. A possible complication to our interpretation of these data is the recent demonstration that Ly can have antiestrogenic activity by binding to ER (Pasapera Limon et al. 2003). Inhibitors also interfered with ERK phosphorylation levels that were not stimulated by xenoestrogens above untreated background levels. For example, endosulfan, which elevates ERK phosphorylation only after 30 min, still participated in a significant lowering of basal ERK phosphorylation levels at early time points. In our previous work (Bulayeva et al. 2004), we demonstrated that these inhibitors by themselves were unable to change basal levels of ERK phosphorylation; the present study thus shows that the presence of xenoestrogens was necessary to produce inhibitor-driven decreases below basal levels. Because ERK phosphorylation demonstrated a complex temporal fluctuation, we speculate that periods of "dephosphorylation" demonstrated by our data could be the result of desensitization of the stimulatory pathways and/or phosphatase activation. Such deactivation and reactivation profiles may be very important for specific estrogenic stimulatory effects because other hormones and regulators are known to operate in an oscillatory fashion through kinase inactivation (MacDonald et al. 1997) or protein degradation (Murray 2004). However, the rapid recovery times in our pattern argue against the latter mechanism.
Wide diversity in signaling cascades leading to ERK activation can perhaps be explained by the nature of mERs and the probable necessity of their interactions with many other different signaling partners. Xenoestrogens are highly diverse in structure, and the conformation of different xenoestrogen-ER complexes could be significantly different from that of an E 2 -ER complex (Brzozowski et al. 1997;Suzuki et al. 2001;van Lipzig et al. 2004). This could alter the receptor protein's surface topography and thus its interactions with partner proteins, as has been demonstrated for ligand effects on nuclear receptor interactions with coactivators and corepressors. The nature and magnitude of responses are probably a function of the conformation the receptor assumes around these diverse molecules and the repertoire of interacting proteins present in different cell types (so pituitary cell patterns may not be predictive for other cell types). The outcomes can be different and multiplex. Therefore, xenoestrogens will need to be individually examined for these complex mechanistic and functional outcomes in different tissues.


s2fieldsofstudy:
Biology
Environmental Science

text:
Flea-borne Bartonella grahamii and Bartonella taylorii in Bank Voles

Bartonella species are increasingly associated with a range of human and animal diseases. Despite this, we have a poor understanding of the ecology and epidemiology of many species, especially those circulating in wild populations. Previous studies have demonstrated that a diverse range of Bartonella species are abundant in wild rodent populations; little is known regarding their modes of transmission, although both direct and indirect routes have been suggested. In this study, with bank voles (Clethrionomys glareolus) as the host species, we demonstrate that the rodent flea Ctenophthalmus nobilis is a competent vector of at least two Bartonella species, B. grahamii, which has previously been associated with human infection, and B. taylorii. In contrast, no evidence of either horizontal or vertical transmission was seen in bank voles inoculated with B. taylorii maintained in an arthropod-free environment; this finding suggests that fleas may be essential for transmitting some Bartonella species.

Bartonella species are increasingly associated with a range of human and animal diseases. Despite this, we have a poor understanding of the ecology and epidemiology of many species, especially those circulating in wild populations. Previous studies have demonstrated that a diverse range of Bartonella species are abundant in wild rodent populations; little is known regarding their modes of transmission, although both direct and indirect routes have been suggested. In this study, with bank voles (Clethrionomys glareolus) as the host species, we demonstrate that the rodent flea Ctenophthalmus nobilis is a competent vector of at least two Bartonella species, B. grahamii, which has previously been associated with human infection, and B. taylorii. In contrast, no evidence of either horizontal or vertical transmission was seen in bank voles inoculated with B. taylorii maintained in an arthropodfree environment; this finding suggests that fleas may be essential for transmitting some Bartonella species.
T he genus Bartonella currently contains 19 species of gram-negative bacteria that parasitize the erythrocytes of vertebrate hosts, and an increasing number of species are now considered as emerging infections of medical and veterinary importance (1). In addition to humans and domesticated animals, they have also been isolated from a variety of wild mammal species, including cervids, ruminants, carnivores, and rodents (1)(2)(3). Of these, rodents are perhaps the best studied, with high prevalences of Bartonella infections, coupled with highly diverse species and strains (4)(5)(6)(7). Human infections with Bartonella species of rodent origin have been reported from both sides of the Atlantic: in the United States, B. elizabethae, associated with endocarditis, B. washoensis, associated with cardiac disease, and B. vinsonii subsp. arupensis, causing fever and neurologic symptoms (8)(9)(10); in Europe, B. grahamii, isolated from the eye of a patient with neuroretinitis (11).
While the association of Bartonella of rodent origin with human disease continues to increase, our understanding of the ecology and epidemiology of these infections is scant. Fundamental to this endeavor would be clarifying their mode(s) of transmission. Bartonella species are generally considered to be transmitted by arthropod vectors, and Bartonella DNA has been detected in fleas and ticks collected from both wild and domestic animals (12)(13)(14)(15)(16). However such findings do not necessarily prove vector competence, and vectors have only been conclusively identified for a few species: the sand fly (Lutzomyia verrucarum) for B. bacilliformis (17), the body louse (Pediculus humanus) for B. quintana (18), and the cat flea (Ctenocephalides felis) for B. henselae (19). Anecdotal evidence exists for the role of ticks as vectors of at least some Bartonellae (20)(21)(22). For rodent Bartonellae, two vectors have been suggested. The oriental rat flea (Xenopsylla cheopis) was demonstrated to be a competent vector of an unidentified Bartonella species that infected bank voles (Clethrionomys glareolus) (23), and the vole ear mite (Trombicula microti) was proposed as a vector of B. vinsonii (24). However, no experimental transmission studies have been undertaken in which the Bartonella species involved could be accurately identified by, for example, using a molecular approach. In addition, vertical transmission has been suggested as a potential mechanism by which infection may be maintained within a population (25), and experimental data suggest that transplacental transmission occurred in BALB/c mice infected with B. birtlesii, although no viable fetuses were bacteremic (26).
The aim of this study was to determine the potential for fleas, collected from a population of bank voles in which Bartonella infections were known to be endemic, to transmit infection to naïve bank voles. In addition, the potential importance of direct horizontal or vertical transmission was investigated.

Materials and Methods.
Twenty fleas were collected from six bank voles sampled in a mixed woodland in northwest England (53°20.6N, 3°02.4W) where previous studies had shown the prevalence of Bartonella infection in bank voles was approximately 60% (5). These fleas were added to a rodent "arena," measuring 1.2 m x 1.2 m in a temperature-

Flea-borne Bartonella grahamii and Bartonella taylorii in Bank Voles
Kevin J. Bown,* Malcolm Bennett,* and Michael Begon* controlled room. The arena contained sawdust as substrate, hay and shredded paper as bedding, and Longworth traps (Abingdon, UK) set on prebait as nest boxes. No Bartonella spp. had been used in experiments in the arena before the introduction of the fleas, and no fleas had previously been kept in the arena. The arena had been kept free of bank voles for 2 weeks before this study began. Twentyeight captive-bred bank voles from a Bartonella-free colony maintained at the University of Liverpool were added to the arena immediately after the fleas were introduced. All of these voles had tested negative for Bartonella infection before entering the arena. Four weeks after the bank voles were added to the arena, all were euthanized, and blood samples were collected by cardiac puncture. In addition to the 28 voles originally introduced to the arena, one female had produced a litter, and the two pups produced were also humanely killed and had sterile blood samples collected. Fleas were collected from all rodents and kept in individual tubes (1 per rodent) containing 70% ethanol. A sample of fleas from bedding within the arena was collected at the same time. All fleas were identified to species level (27).
Isolation of Bartonella spp. from the blood samples was undertaken by plating freeze-thawed blood onto Colombia blood agar plates enriched with 5% horse blood. Plates were incubated at 37°C and 5% CO 2 for up to 14 days. Isolates putatively resembling Bartonella spp. colonies were further characterized by polymerase chain reaction (PCR). Individual colonies were prepared by boiling in 100 µL of sterile deionized distilled water for 10 min. Five microliters of this preparation were used as template. Each 50-µL reaction contained 1.25 U of Taq polymerase, 200 mmol of each dNTP, 1.5 mmol of MgCl 2 , and 30 pmol of each primer. Initial characterization used primers QHVE1 and QHVE3 (28) that target the 16S-23S rRNA intergenic spacer region. PCR products from positive samples were purified by using the Promega Wizard PCR Preps kit (Promega, Madison, WI), and then digested with HaeIII as previously described (29). Samples relating to each REA pattern were then analyzed by using primers BhCS781.p and BhCS1137.n (30), which target the citrate synthase (gltA) gene. After purification these PCR products were sequenced with an ABI 377 automated sequencer, and the sequences were compared with previously published sequences by using the BLAST program from the National Center for Biotechnology Information Web site (available from: http:/www.ncbi.nlm.nih.gov/ BLAST/).
Horizontal and vertical transmission experiments were undertaken using 16 bank voles, housed, in the absence of fleas, in cages containing male-female pairs approximately 4 weeks of age, one or both of which was injected through the footpad with approximately 10 6 CFU of Bartonella taylorii. In two of the cages both voles were inoculated, in two only the male vole was inoculated, and in four others only the female vole was inoculated. Pairs were kept until they had produced a litter. Blood samples were taken at day 0, when the voles were inoculated, at day 10 to confirm infection status of the adults, and 8 weeks later when litters were between 7 and 14 days old. Isolation attempts were carried out as described.

Results
Twenty-one of the 28 blood samples from the bank voles produced colonies resembling Bartonella spp., and all of these were confirmed as Bartonella spp. by PCR. Restriction enzyme analysis of the resulting PCR products showed that two different Bartonella genotypes were present in the bank voles ( Figure). Sequence analysis of the gltA gene showed these to represent B. taylorii (16 isolates) and B. grahamii (6 isolates) (one bank vole was coinfected with both). In addition to the original 28 voles added to the arena, two pups were sampled that had been suckling from a bacteremic female. Neither was bacteremic.
A total of 217 fleas were collected from the 28 bank voles (mean 7.75 fleas per vole). Only one species of flea was identified, Ctenophthalmus nobilis nobilis. Ten pools of five randomly selected fleas collected from the voles were tested for Bartonella spp. DNA using the gltA PCR. its mate, despite that all inoculated animals remained bacteremic throughout the experiment. Seven of the eight pairs of voles produced a litter, one of the pairs in which the female alone was inoculated did not. A total of 20 young were produced from the seven litters, with litter sizes ranging between one and five offspring (mean 2.86 offspring per litter). No bacteremia could be detected in any of the offspring, whether only one or both parents had been inoculated.

Discussion
This study shows that fleas are efficient vectors of at least some rodent bartonellae. Twenty one of 28 (75%) naïve bank voles housed with wild-caught fleas for 4 weeks became bacteremic, 16 voles (57.1%) infected with B. taylorii and 6 voles (21.4%) infected with B. grahamii. Similarly, each of 10 pools of 5 fleas collected showed the presence of Bartonella spp. DNA within them, and 7 of 10 individual fleas were also positive. Fleas have previously been implicated in the transmission of B. henselae infections of cats (19,31,32), and Bartonella DNA has previously been detected in fleas collected from rodents (14,15), but no recent experimental studies on the role of fleas in the transmission of rodent bartonellae have been reported since early studies by Krampitz (23) indicated that fleas could transmit an unidentified Bartonella species. In fact, two different Bartonella species could be transmitted by a single species of flea, suggesting little vector-bacteria specificity.
On the other hand, no transmission occurred between infected and susceptible animals when housed together in the same cage in the absence of fleas, and no transmission could be detected from parent to offspring, although larger numbers of animals may be needed to confirm that such transmission does not occur. This absence of vertical transmission agrees with results of a study of cats infected with B. henselae (33,34), but Kosoy and colleagues (25) found that Bartonella could be isolated from the neonates and embryos of naturally infected North American rodents, while transplacental transmission of B. birtlesii was also reported in BALB/c mice, although none of the viable offspring in that study were bacteremic (26).
These findings are of potential public health importance: B. grahamii has previously been associated with human disease (11), although the pathogenic potential of B. taylorii is as yet unknown. Furthermore, the implication of fleas in the transmission of these rodent Bartonella, as well as B. henselae (19,31) suggests that fleas may be involved in the transmission of many other rodent Bartonella species, some of which have already been shown to be pathogenic to humans. Whether the route of rodent to human transmission is likely to be due to flea transmission or through direct contact, such as bites or scratches as is commonly the case for B. henselae (1), needs to be investigated.
The exact route by which fleas transmitted Bartonella to susceptible rodents remains unclear. Future work should seek to distinguish the role of fecal contamination and then the role of scratching (32) from direct transmission through feeding. Investigating the efficiency of different flea species in transmitting a variety of Bartonella species would be valuable as would determining whether fleas infected with a number of Bartonella species transmit one species more efficiently than the others. Studies such as these would help expand the current knowledge on vector-Bartonella specificity and determine its importance in influencing the diversity of Bartonella species.
Dr. Bown is currently a research associate in the Faculty of Veterinary Science at the University of Liverpool. His interests focus on the ecology of wildlife diseases, particularly those caused by vector-borne bacteria.

Flea-borne Bartonella Infections in Bank Voles
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