Calcium binding to the chloroplast andE. coli (CF0) F0 subunit III (c) of the ATP-synthase

Summary Subunit III and c, the 8 kDa components of the chloroplast CF₀, andE. coli H⁺ channel complexes respectively, were isolated and purified for the purpose of studying their Ca⁺⁺-binding properties. Purified subunit III or c as well as the unfractionated organic-solvent soluble preparation from chloroplasts were used in a⁴⁵Ca⁺⁺-ligand blot assay known to detect high affinity Ca⁺⁺-binding sites in proteins. Both subunit III and c showed strong⁴⁵Ca⁺⁺-binding. None of the other CF₀ subunits bound Ca⁺⁺ and of the CF₁ only a weak binding was detected in the region of the , subunits. The Ca⁺⁺-binding was inhibited after treating the proteins in solution by derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La³⁺. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu 61 (Asp 61 inE. coli), did not inhibit the Ca⁺⁺-binding in either protein. The data indicate that for both proteins the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III (or c) C-terminal provide some of seven essential oxygen ligands normally required for defining a Ca⁺⁺-binding site in proteins. Based on the accepted models for the hairpin conformation of the subunit III (c), it seems clear that the Ca⁺⁺-binding site can form on the lumenal side of the membrane in the functional CF₀ structures or on the periplasmic side of theE. coli membrane. A working hypothesis we are testing is that Ca⁺⁺-binding to the CF₀ (or F₀) can form an easily reversible gating site such as to enhance the probability for membrane-localized H⁺ gradients being coupled to ATP formation under moderate energization loads, but under excess energization the local H⁺ ion concentration may build up high enough to displace the bound Ca⁺⁺, resulting in delocalization of the H⁺ gradient. The latter situation seems, in chloroplasts at least, to function as a signal for over-energization; i.e., excess light absorption, a potential stress situation for plants. Lumenal acidification appears to be a trigger for initiating stress alleviation responses.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Sciences, Puschchino, Russia.     

Mitochondrial F(0)F(1) ATP synthase. Subunit regions on the F1 motor shielded by F(0), Functional significance, and evidence for an involvement of the unique F(0) subunit F(6)

Studies reported here were undertaken to gain greater molecular insight into the complex structure of mitochondrial ATP synthase (F(0)F(1)) and its relationship to the enzyme's function and motor-related properties. Significantly, these studies, which employed N-terminal sequence, mass spectral, proteolytic, immunological, and functional analyses, led to the following novel findings. First, at the top of F(1) within F(0)F(1), all six N-terminal regions derived from alpha + beta subunits are shielded, indicating that one or more F(0) subunits forms a "cap." Second, at the bottom of F(1) within F(0)F(1), the N-terminal region of the single delta subunit and the C-terminal regions of all three alpha subunits are shielded also by F(0). Third, and in contrast, part of the gamma subunit located at the bottom of F(1) is already shielded in F(1), indicating that there is a preferential propensity for interaction with other F(1) subunits, most likely delta and epsilon. Fourth, and consistent with the first two conclusions above that specific regions at the top and bottom of F(1) are shielded by F(0), further proteolytic shaving of alpha and beta subunits at these locations eliminates the capacity of F(1) to couple a proton gradient to ATP synthesis. Finally, evidence was obtained that the F(0) subunit called "F(6)," unique to animal ATP synthases, is involved in shielding F(1). The significance of the studies reported here, in relation to current views about ATP synthase structure and function in animal mitochondria, is discussed.     

Mutations in the conserved proline 43 residue of the uncE protein (subunit c) of Escherichia coli F1F0-ATPase alter the coupling of F1 to F0

The conserved Pro43 residue of the uncE protein (subunit c) of the Escherichia coli F1F0-ATPase was changed to Ser or Ala by oligonucleotide-directed mutagenesis, and the mutations were incorporated into the chromosome. The resultant mutant strains were capable of oxidative phosphorylation as indicated by their ability to grow on succinate and had growth yields on glucose that were 80-90% of wild type. Membrane vesicles from the mutants were slightly less efficient than wild type vesicles in ATP-driven proton pumping as indicated by ATP-dependent quenching of quinacrine fluorescence. The decreased quenching response was not due to increased H+ leakiness of the mutant membranes or to loss of F1-ATPase activity from the membrane. These results indicate that the mutant F1F0-ATPases are defective in coupling ATP hydrolysis to H+ translocation. The membrane ATPase activity of the mutants was inhibited less by dicyclohexylcarbodiimide than that of wild type. The decrease in sensitivity to inhibition by dicyclohexylcarbodiimide was caused primarily by dissociation of the F1-ATPase from the mutant F0 in the ATPase assay mixture. These results support the idea that Pro43, and neighboring conserved polar residues play an important role in the binding and functional coupling of F1 to F0. Although a Pro residue is found at position 43 in all species of subunit c studied, surprisingly, it is not absolutely essential to function.     

F(1)F(0) ATP synthase subunit c is targeted by the SRP to YidC in the E. coli inner membrane

Escherichia coli inner membrane proteins (IMPs) use different pathways for targeting and membrane integration. We have examined the biogenesis of the F1F0 ATP synthase subunit c, a small double spanning IMP, using complementary in vivo and in vitro approaches. The data suggest that F0c is targeted by the SRP to the membrane, where it inserts at YidC in a Sec-independent mechanism. F0c appears to be the first natural substrate of this novel pathway.     

YidC-mediated membrane insertion of assembly mutants of subunit c of the F1F0 ATPase

YidC is a member of the OxaI family of membrane proteins that has been implicated in the membrane insertion of inner membrane proteins in Escherichia coli. We have recently demonstrated that proteoliposomes containing only YidC support both the stable membrane insertion and the oligomerization of the c subunit of the F(1)F(0) ATP synthase (F(0)c). Here we have shown that two mutants of F(0)c unable to form a functional F(1)F(0) ATPase interact with YidC, require YidC for membrane insertion, but fail to oligomerize. These data show that oligomerization is not essential for the stable YidC-dependent membrane insertion of F(0)c consistent with a function of YidC as a membrane protein insertase.     

H+-ATPase activity of Escherichia coli F1F0 is blocked after reaction of dicyclohexylcarbodiimide with a single proteolipid (subunit c) of the F0 complex

Dicyclohexylcarbodiimide (DCCD) specifically inhibits the F1F0-H+-ATP synthase complex of Escherichia coli by covalently modifying a proteolipid subunit that is embedded in the membrane. Multiple copies of the DCCD-reactive protein, also known as subunit c, are found in the F1F0 complex. In order to determine the minimum stoichiometry of reaction, we have treated E. coli membranes with DCCD, at varying concentrations and for varying times, and correlated inhibition of ATPase activity with the degree of modification of subunit c. Subunit c was purified from the membrane, and the degree of modification was determined by two methods. In the "specific radioactivity" method, the moles of [14C]DCCD per total mole of subunit c was calculated from the radioactivity incorporated per mg of protein, and conversion of mg of protein to mol of protein based upon amino acid analysis. In the "high performance liquid chromatography (HPLC) peak area" method, the DCCD-modified subunit c was separated from unmodified subunit c on an anion exchange AX300 HPLC column, and the areas of the peaks from the chromatogram quantitated. The shape of the modification versus inhibition curve indicated that modification of a single subunit c per F0 was sufficient to abolish ATPase activity. The titration data were fit by nonlinear regression analysis to a single hit mathematical model, A = Un(1 - r) + r, where A is the relative activity, U is the ratio of unmodified/total subunit c, n is the number of subunit c per F0, and r is a residual fraction of ATPase activity that was resistant to inhibition by DCCD. The two methods gave values for n equal to 10 by the specific radioactivity method and 14 by the HPLC peak area method, and values for r of 0.28 and 0.30, respectively. Most of the r value was accounted for by the observed dissociation of 15-20% of the F1-ATPase from the membrane under ATPase assay conditions. When the minimal, experimentally justified value of r = 0.15 was used in the equation above, the calculated values of n were reduced to 8 and 11, respectively. The value of n determined here, with a probable range of uncertainty of 8-14, is consistent with, and provides an independent type of experimental support for, the suggested stoichiometry of 10 +/- 1 subunit c per F1F0, which was determined by a more precise radiolabeling method (Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009-2015).     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.     

Localization of the delta subunit in the Escherichia coli F(1)F(0)-ATPsynthase by immuno electron microscopy: the delta subunit binds on top of the F(1)

The binding site of the delta subunit in the F(1)F(0)-ATPsynthase from Escherichia coli has been determined by electron microscopy of negatively stained, antibody-decorated enzyme molecules. The images show that the antibody is bound at the very top of the F(1) domain indicating that at least part of delta is bound in the dimple formed by the N termini of the alpha and beta subunits. The data may explain why there is only one binding site for delta on the F(1) despite there being three identical alphabeta pairs. The finding also implies that the b subunits of the F(0) have to extend all the way from the membrane surface to the very top of the F(1) domain to make contact with the delta subunit.     

Mutagenesis mapping of the presenilin 1 calcium leak conductance pore

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca(2+) leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca(2+) conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca(2+) leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca(2+) conductance pore.     

The dimerization domain of the b subunit of the Escherichia coli F(1)F(0)-ATPase.

In this study a series of N- and/or C-terminal truncations of the cytoplasmic domain of the b subunit of the Escherichia coli F(1)F(0) ATP synthase were tested for their ability to form dimers using sedimentation equilibrium ultracentrifugation. The deletion of residues between positions 53 and 122 resulted in a strongly decreased tendency to form dimers, whereas all the polypeptides that included that sequence exhibited high levels of dimer formation. b dimers existed in a reversible monomer-dimer equilibrium and when mixed with other b truncations formed heterodimers efficiently, provided both constructs included the 53-122 sequence. Sedimentation velocity and (15)N NMR relaxation measurements indicated that the dimerization region is highly extended in solution, consistent with an elongated second stalk structure. A cysteine introduced at position 105 was found to readily form intersubunit disulfides, whereas other single cysteines at positions 103-110 failed to form disulfides either with the identical mutant or when mixed with the other 103-110 cysteine mutants. These studies establish that the b subunit dimer depends on interactions that occur between residues in the 53-122 sequence and that the two subunits are oriented in a highly specific manner at the dimer interface.     

Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase

Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H(+)-transporting ATPases. The c(2) and c(3) oligomers have proven useful in testing hypothesis regarding the mechanism of energy coupling. In the first part of this paper, the uncoupling Q42E substitution has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer. Both mutant proteins proved to be as functional as the wild type c(2) dimer or wild type c(3) trimer. The results argue against an obligatory movement of the epsilon subunit between loops of monomeric subunit c in the c(12) oligomer during rotary catalysis. Rather, the results support the hypothesis that the c-epsilon connection remains fixed as the c-oligomer rotates. In the second section of this paper, we report on the effect of substitution of the proton translocating Asp(61) in every second helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer. Based upon the precedent of V-type ATPases, where the c(2) dimer occurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E. coli c(2) and c(3) fused proteins were predicted to have a functional H(+)-transporting ATPase activity, with a reduced H(+)/ATP stoichiometry, but to be inactive as ATP synthases. A variety of Asp(61)-substituted proteins proved to lack either activity indicating that the switch in function in V-type ATPases is a consequence of more than a single substitution.     

Opposing actions of angiotensins on angiogenesis

Using the murine sponge model of angiogenesis, associated to functional and morphological parameters we have demonstrated opposing actions of angiotensin II (Ang II) and angiotensin-(1-7;Ang-1-7) in modulating fibrovascular tissue growth. Angiogenesis in the implants was assessed at day 7 postimplantation by extracting the hemoglobin content, by determining the outflow rate of sodium fluorescein applied intraimplant and by histological analysis. Furthermore, the proliferative activity of control and angiotensin-treated implants was established using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4 -sulfonyl)2H-tetrazolium)assay. The hemoglobin content in the control implants was 2.4 +/- 0.14 (microg/mg wet weight) versus 3.6 +/- 0.27(Ang II;100 ng) and 0.86 +/- 0.07 Ang-(1-7); 20 ng. Blood flow in the implants as determined by t1/2 values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation) showed that Ang II stimulated angiogenesis, whereas Ang-(1-7) inhibited it. The proliferative activity of the sponge-induced fibrovascular tissue was enhanced by Ang II and diminished by Ang-(1-7). These results show the pro-versus anti-angiogenic effects of these angiotensin molecules, providing evidence for their opposing effects on vascular tissue growth and wound healing in vivo.     

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251-255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1-113) and ANX-I(34-346) were fully active, whereas ANX-I(114-346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.     

Mutational analysis of the glycine-rich region of the c subunit of the Escherichia coli F0F1 ATPase.

Eight strains carrying amino acid substitutions within the c subunit of the F0F1 ATPase of Escherichia coli have been constructed by using site-directed mutagenesis. Three strains carrying the substitutions Gly-23----Leu, Ala-24----Leu, and Gly-38----Leu, which reside in or near the highly conserved glycine-rich region of the c subunit, are unable to carry out oxidative phosphorylation. Membranes prepared from these strains possess basal levels of ATPase activity. In contrast, strains carrying the substitutions Ile-30----Phe, Gly-33----Leu, Gly-58----Leu, and Lys-34----Val and the Lys-34----Val, Glu-37----Gln double substitution were found to possess a coupled phenotype similar to that of the wild type.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane

Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase. Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis. Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain. While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes. Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.     

Defining the domain of binding of F1 subunit epsilon with the polar loop of F0 subunit c in the Escherichia coli ATP synthase.

We have previously shown that the E31C-substituted epsilon subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 24609-24614). The interactions of subunits epsilon and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1. To further define the domains of interaction, we have introduced additional Cys into subunit epsilon and subunit c and tested for cross-link formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon, can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c. The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits. In the model, residues 26-33 form a turn of antiparallel beta-sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer.     

Rotation of the c subunit oligomer in EF(0)EF(1) mutant cD61N

ATP synthases (F(0)F(1)-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F(0), to ATP synthesis within the peripheral portion, F(1). The coupling most probably occurs through the rotation of a central rotor (subunits c(10)epsilon gamma) relative to the stator (subunits ab(2)delta(alpha beta)(3)). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab(2). In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF(0)F(1) and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit gamma, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Side-chain charge effects and conductance determinants in the pore of ClC-0 chloride channels

The charge on the side chain of the internal pore residue lysine 519 (K519) of the Torpedo ClC-0 chloride (Cl-) channel affects channel conductance. Experiments that replace wild-type (WT) lysine with neutral or negatively charged residues or that modify the K519C mutant with various methane thiosulfonate (MTS) reagents show that the conductance of the channel decreases when the charge at position 519 is made more negative. This charge effect on the channel conductance diminishes in the presence of a high intracellular Cl- concentration ([Cl-]i). However, the application of high concentrations of nonpermeant ions, such as glutamate or sulfate (SO42-), does not change the conductance, suggesting that the electrostatic effects created by the charge at position 519 are unlikely due to a surface charge mechanism. Another pore residue, glutamate 127 (E127), plays an even more critical role in controlling channel conductance. This negatively charged residue, based on the structures of the homologous bacterial ClC channels, lies 4-5 A from K519. Altering the charge of this residue can influence the apparent Cl- affinity as well as the saturated pore conductance in the conductance-Cl- activity curve. Amino acid residues at the selectivity filter also control the pore conductance but mutating these residues mainly affects the maximal pore conductance. These results suggest at least two different conductance determinants in the pore of ClC-0, consistent with the most recent crystal structure of the bacterial ClC channel solved to 2.5 A, in which multiple Cl--binding sites were identified in the pore. Thus, we suggest that the occupancy of the internal Cl--binding site is directly controlled by the charged residues located at the inner pore mouth. On the other hand, the Cl--binding site at the selectivity filter controls the exit rate of Cl- and therefore determines the maximal channel conductance.