Structure-function relationships in membrane segment 5 of the yeast Pma1 H+-ATPase

Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

Effect of N-palmitoylethanolamine on energy-dependent transport of Ca2+ in vesicles of myometrium sarcolemma and their phospholipid composition

N-palmitoylethanolamine (NPE) was studied for their effect on calcium pump of pig myometrium sarcolemma. NPE in concentration of 10 microM, stimulated by 28-46% Mg2+, ATP-dependent accumulation of Ca2+ in vesicles of plasmatic membrane of uterus myocytes taking absolutely no effect on passive release of this cation from them. NPE modified phospholipid composition of sarcolemma, causing the increase of percentage content of phosphatidylinositol (by 20.2%) and lysophosphatidylcholine (2.7 times). While NPE effects transport Ca2+, Mg(2+)-ATPase solubilized from plasmatic membrane and purified due to the method of affinity chromatography on calmodulin-sepharose 4B, no activating effect of NPE on the calcium pump was observed. And what is more, a weakly expressed tendency to inhibition (by 14-15%, respectively) of the rate of Ca2+, Mg(2+)-dependent enzymic hydrolysis of ATP has been revealed. It is supposed that the effect of NPE on active transmembrane transport of Ca2+ is an important link in the general mechanism of contraction-relax of the myometrium and is, apparently, connected with its modifying effect on the lipid composition of the sarcolemma.     

Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin

The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.     

The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase

During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.     

Spectroscopic studies of the interaction of Ca2+-ATPase-peptides with dodecyl maltoside and its brominated analog

The transmembrane sector of sarcoplasmic reticulum Ca2+-ATPase comprises ten putative transmembrane spans (M1-M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding: peptide M6 (amino acid residues 785-810), peptide M7-L (amino acid residues 808-847) corresponding to loop 6-7 and the majority of span M7, and peptide M7-S (amino acid residues 818-847) which contains a shorter version of loop 6-7 than M7-L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the alpha-helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7-L peptide, the L6-7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+-ATPase. The formation of the peptide-detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n-dodecyl beta-D-maltoside or quenching using the recently introduced brominated analog of n-dodecyl beta-D-maltoside: 7,8-dibromododecyl beta-maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur J. Biochem. 241, 343-354]. Our results indicate that M7-L and M7-S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size-exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n-dodecyl beta-D-maltoside/7,8-dibromododecyl beta-maltoside, using tryptophan octyl ester and solubilized Ca2+-ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+-ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+-ATPase and the rather short alpha-helix span of M6 may lead to exposure of a significant part of the C-terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site-directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.     

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.     

A new topological model of the cardiac sarcolemmal Na+-Ca2+ exchanger

The current topological model of the Na+-Ca2+ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na+-Ca2+ exchanger. This mutant is fully functional in Na+ gradient-dependent 45Ca2+ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.     

Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger

The cardiac Na+/Ca2+ exchanger, NCX1, has been modeled to consist of 11 transmembrane segments and a large cytoplasmic loop (loop f). Cysteine mutagenesis and sulfhydryl modification experiments demonstrate that the loop connecting transmembrane segments 1 and 2 (loop b) is located on the cytoplasmic side of the membrane, as previously modeled. A mutation in loop b, asparagine 101 to cysteine (N101C), renders the exchanger insensitive to regulation by cytoplasmic Na+ and Ca2+. Nearby mutations at residue threonine 103 (T103C or T103V) increase the apparent affinity of the exchanger for cytoplasmic Na+ and also produce a significant Li+ transport capacity. The evidence suggests that the region at the interface of cytoplasmic loop b and transmembrane segment 2 is important in Na+ transport and also in secondary regulation. Thus, this region may form part of the link between the ion translocation pathway formed by the transmembrane segments and regulatory sites that have previously been localized to loop f.     

Location of the membrane-docking face on the Ca2+-activated C2 domain of cytosolic phospholipase A2

Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.     

Local anesthetic block of batrachotoxin-resistant muscle Na+ channels

Local anesthetics (LAs) are noncompetitive antagonists of batrachotoxin (BTX) in voltage-gated Na+ channels. The putative LA receptor has been delineated within the transmembrane segment S6 in domain IV of voltage-gated Na+ channels, whereas the putative BTX receptor is within segment S6 in domain I. In this study, we created BTX-resistant muscle Na+ channels at segment I-S6 (micro1-N434K, micro1-L437K) to test whether these residues modulate LA binding. These mutant channels were expressed in transiently transfected human embryonic kidney 293T cells, and their sensitivity to lidocaine, QX-314, etidocaine, and benzocaine was assayed under whole-cell, voltage-clamp conditions. Our results show that LA binding in BTX-resistant micro1 Na+ channels was reduced significantly. At -100 mV holding potential, the reduction in LA affinity was maximal for QX-314 (by 17-fold) and much less for neutral benzocaine (by 2-fold). Furthermore, this reduction was residue specific; substitution of positively charged lysine with negatively charged aspartic acid (micro1-N434D) restored or even enhanced the LA affinity. We conclude that micro1-N434K and micro1-L437K residues located near the middle of the I-S6 segment of Na+ channels can reduce the LA binding affinity without BTX. Thus, this reduction of the LA affinity by point mutations at the BTX binding site is not caused by gating changes induced by BTX alone. We surmise that the BTX receptor and the LA receptor within segments I-S6 and IV-S6, respectively, may align near or within the Na+ permeation pathway.     

Oligomerization and divalent ion binding properties of the S100P protein: a Ca2+/Mg2+-switch model

S100P is a 95 amino acid residue protein which belongs to the S100 family of proteins containing two putative EF-hand Ca2+-binding motifs. In order to characterize conformational properties of S100P in the presence and absence of divalent cations (Ca2+, Mg2+ and Zn2+) in solution, we have analyzed hydrodynamic and spectroscopic characteristics of wild-type and several variants (Y18F, Y88F and C85S) of S100P using equilibrium centrifugation, gel-filtration chromatography, circular dichroism and fluorescence spectroscopies. Analysis of the experimental data shows the following. (1) In agreement with the predictions there are two Ca2+-binding sites in the S100P molecule with different affinity; the high affinity binding site has an apparent binding constant of approximately 10(7) M-1 and the low affinity binding site has an apparent binding constant of approximately 10(4) M-1. (2) The high and low affinity Ca2+-binding sites are located in the C and N-terminal parts of the S100P molecule, respectively. (3) These C and N-terminal sites can also bind other divalent ions. The C-terminal site binds Zn2+ (with relatively low affinity approximately 10(3) M-1), but not Mg2+. The N-terminal site binds Mg2+ with the apparent binding constant approximately 10(2) M-1. (4) Binding of Ca2+ to the C-terminal site and binding of Mg2+ to the N-terminal site occur in the physiological concentration range of these ions (micromolar for Ca2+ and millimolar for Mg2+). (5) Oligomerization state of the S100P molecule appears to change upon addition of Ca2+. On the basis of these observations a plausible model for S100P as a Ca2+/Mg2+ switch has been proposed.     

A randomized controlled study comparing elemental diet and steroid treatment in Crohn''s disease

To elucidate the mechanism underlying the interaction between the L-type Ca2+ channel and the dihydropyridines (DHPs), contribution of the repeat III was studied by constructing chimeras between the DHP-sensitive alpha1C and DHP-insensitive alpha1E subunits. The chimeras were transiently expressed in human embryonic kidney 293 cells and the whole-cell Ba2+ current (IBa) was recorded. Mutating Thr1061 to Tyr in IIIS5 of the alpha1C sequence completely abolished the inhibition and stimulation of IBa by the antagonist (+)-isradipine and agonist (-)-Bay K 8644, whereas mutating Gln1065 to Met in IIIS5 decreased the affinity for isradipine 100-fold without affecting the stimulating effect of Bay K 8644. The conserved amino acid residue Tyr1174 in IIIS6 of the alpha1C subunit was necessary for the high affinity DHP block. The DHP-dependent block and stimulation of IBa were transferred to the alpha1E channel by the mutation of two amino acid residues in IIIS5 (Y1295T, M1299Q), three residues in IIIS6 (F1406I, F1409I, V1414M) and three residues in IVS6 (I1706Y, F1707M, L1714I). The mutated alpha1E channel was stimulated 2.8-fold by 1 microM Bay K 8644 and blocked by isradipine with an IC50 value of 60 nM. These results show that mutation of Thr1061 in the alpha1C sequence results in a DHP-insensitive L-type channel and that transfer of the high affinity DHP sensitivity requires mutation of eight amino acid residues in the alpha1E sequence.     

Change in conformation of plasma membrane phospholipid scramblase induced by occupancy of its Ca2+ binding site

Phospholipid (PL) scramblase is a 35.1 kDa plasma membrane protein that mediates the accelerated transbilayer migration of plasma membrane PL in activated, injured, or apoptotic cells exposed to elevated intracellular Ca2+. We recently identified a conserved segment in the PL scramblase polypeptide (residues Asp273 to Asp284) that is essential for its PL-mobilizing function and was presumed to contain the Ca2+ binding site of the protein (Zhou, Q., Sims, P. J., and Wiedmer, T. (1998) Biochemistry 37, 2356-2360). Whereas the sequence of this peptide segment resembles that of known Ca2+-binding loops within EF-hand containing proteins, it is unusual in being a single such loop in the entire protein and in being closely spaced to the predicted transmembrane helix (Ala291-Gly309). To gain insight into how Ca2+ activates the PL-mobilizing function of PL scramblase, we analyzed conformational changes associated with occupancy of this putative Ca2+ binding site. In addition to activation by Ca2+, the PL-mobilizing function of PL scramblase was found to be activated by other ions, with apparent affinities Tb3+, La3+ > Ca2+ > Mn2+ > Zn2+ > Sr2+ > Ba2+, Mg2+. Evidence for coordinate binding of metal ion by the polypeptide was provided by resonance energy transfer from protein Trp to Tb3+, which was competed by excess Ca2+. Metal binding to PL scramblase was accompanied by increased right-angle light scattering and by a prominent change in circular dichroism, suggesting that coordinate binding of the metal ion induces a conformational change that includes self-aggregation of the polypeptide. Consistent with this interpretation, addition of Ca2+ was found to protect PL scramblase from proteolysis by trypsin both in detergent solution as well as in situ, within the erythrocyte membrane. Mutation in the segment Asp273-Asp284 reduced Tb3+ incorporation and attenuated the change in CD spectrum induced by bound metal ligand, confirming that this suspected EF-hand loopike segment of the polypeptide directly contributes to the Ca2+ binding site.     

Role of the S3 stalk segment in the thapsigargin concentration dependence of sarco-endoplasmic reticulum Ca2+ ATPase inhibition

The sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) is specifically inhibited by thapsigargin (TG), whereas the Na+,K+-ATPase is not. Large chimeric exchanges between Ca2+ and Na+,K+-ATPases (Norregaard, A., Vilsen, B., and Andersen, J. P. (1994) J. Biol. Chem. 269, 26598-26601), as well as photolabeling with a TG azido derivative (Hua, S., and Inesi, G. (1997) Biochemistry 36, 11865-11872), suggest that the S3-M3 (stalk and membrane-bound) region of the Ca2+ ATPase is involved in TG binding. We produced small site-directed changes in the S3 stalk segment of the Ca2+ ATPase and found that mutation of five amino acids to the corresponding Na+,K+-ATPase residues increases by 3 orders of magnitude the TG concentration required for inhibition of Ca2+ ATPase and coupled Ca2+ transport. A single mutation in the S3 stalk segment (Gly257 --> Ile) is sufficient to increase by 1 order of magnitude the TG concentration required to produce 50% inhibition. By comparison, mutations yielding a nine-amino acid homology in the M3 transmembrane segment, or a 25-amino acid homology in the S4 stalk segment, do not affect the ATPase sensitivity to TG. We suggest that specific binding of TG to the S3 stalk segment, in addition to stacking of the TG ring structure at the membrane interface, determines the high affinity of the ATPase for the inhibitor.     

Regulation of cellular Mg2+ by Saccharomyces cerevisiae

Regulation of cellular Mg2+ by S. cerevisiae was investigated. The minimal concentration of Mg2+ that results in optimal growth of S. cerevisiae is about 30 microM and a half-maximum growth rate is attained at about 5 microM Mg2+. Since the plasma membrane has an electrical potential greater than 100 mV, passive equilibration of Mg2+ across the plasma membrane would provide sufficient cytosolic Mg2+ (0.1-1 mM). The total cellular Mg2+ of cells grown in synthetic medium containing 1 mM Mg2+ is about 400 nmol/mg protein, most of which is bound to polyphosphate, nucleic acids, and ATP. Total cellular Mg2+ decreases to about 80 nmol/mg protein as the Mg2+ in synthetic growth medium is reduced to 0.02 mM, but remains relatively constant in growth medium containing 1 to 100 mM Mg2+. Cells shifted into Mg(2+)-free medium continue to grow by utilizing the vacuolar Mg2+ stores. Mg(2+)-starved cells replenish vacuolar Mg2+ stores with a halftime of 30 min. following the addition of 1 mM Mg2+ to the growth medium. The data indicate that cytosolic Mg2+ is maintained by the regulation of Mg2+ fluxes across both the vacuolar and plasma membranes.     

Binding of Ca2+ and Zn2+ to human nuclear S100A2 and mutant proteins

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.     

Role of aromatic transmembrane residues of the delta-opioid receptor in ligand recognition

In the present study we examine the role of transmembrane aromatic residues of the delta-opioid receptor in ligand recognition. Three-dimensional computer modeling of the receptor allowed to identify an aromatic pocket within the helices bundle which spans transmembrane domains (Tms) III to VII and consists of tyrosine, phenylalanine, and tryptophan residues. Their contribution to opioid binding was assessed by single amino acid replacement: Y129F and Y129A (Tm III), W173A (Tm IV), F218A and F222A (Tm V), W274A (Tm VI), and Y308F (Tm VII). Scatchard analysis shows that mutant receptors, transfected into COS cells, are expressed at levels comparable with that of the wild-type receptor. Binding properties of a set of representative opioids were examined. Mutations at position 129 most dramatically affected the binding of all tested ligands (up to 430-fold decrease of deltorphin II binding at Y129A), with distinct implication of the hydroxyl group and the aromatic ring, depending on the ligand under study. Affinity of most ligands was also reduced at Y308F mutant (up to 10-fold). Tryptophan residues seemed implicated in the recognition of specific ligand classes, with reduced binding for endogenous peptides at W173A mutant (up to 40-fold) and for nonselective alkaloids at W274A mutant (up to 65-fold). Phenylalanine residues in Tm V appeared poorly involved in opioid binding as compared with other aromatic amino acids examined. Generally, the binding of highly selective delta ligands (TIPPpsi, naltrindole, and BW373U86) was weakly modified by these mutations. Noticeably, TIPPpsi binding was enhanced at W274A receptor by 5-fold. Conclusions from our study are: (i) aromatic amino acid residues identified by the model contribute to ligand recognition, with a preponderant role of Y129; (ii) these residues, which are conserved across opioid receptor subtypes, may be part of a general opioid binding domain; (iii) each ligand-receptor interaction is unique, as demonstrated by the specific binding pattern observed for each tested opioid compound.     

Tryptophan 388 in putative transmembrane segment 10 of the rat glucose transporter Glut1 is essential for glucose transport

The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr446 and Trp455 in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr446 was found to be essential in that its replacement by any of the other 19 amino acids abolished transport activity (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). The Glut1 glucose transporter of animal cells belongs to the same Glut transporter family as does Gal2 and thus might be expected to show a similar mechanism of substrate recognition. The role of the two amino acids, Phe379 and Trp388, in rat Glut1 corresponding to Tyr446 and Trp455 of Gal2 was therefore studied. Phe379 and Trp388 were individually replaced with each of the other 19 amino acids, and the mutant Glut1 transporters were expressed in yeast. The expression level of most mutants was similar to that of the wild-type Glut1, as revealed by immunoblot analysis. Glucose transport activity was assessed by reconstituting a crude membrane fraction of the yeast cells in liposomes. No significant glucose transport activity was observed with any of Trp388 mutants, whereas the Phe379 mutants showed reduced or no activity. These results indicate that the two aromatic amino acids in TM10 of Glut1 are important for glucose transport. However, unlike Gal2, the residue at the cytoplasmic end of TM10 (Trp388, corresponding to Trp455 of Gal2), rather than that in the middle of TM10 (Phe379, corresponding to Tyr446 of Gal2), is essential for transport activity.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)