Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels

Phosphatidylinositol 4,5-bisphosphate (PIP₂) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca²⁺- and voltage-dependent K⁺ (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP₂ on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP₂ inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP₂ markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2–19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP₂ promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP₂ augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP₂ is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP₂ may exert distinct tissue- and divalent cation–dependent modulatory influences.     

Essential Role of the EF-hand Domain in Targeting Sperm Phospholipase Cζ to Membrane Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Sperm-specific phospholipase C-ζ (PLCζ) is widely considered to be the physiological stimulus that triggers intracellular Ca²⁺ oscillations and egg activation during mammalian fertilization. Although PLCζ is structurally similar to PLCδ1, it lacks a pleckstrin homology domain, and it remains unclear how PLCζ targets its phosphatidylinositol 4,5-bisphosphate (PIP₂) membrane substrate. Recently, the PLCδ1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLCδ1 are notably conserved in PLCζ. We investigated the potential role of these conserved cationic residues in PLCζ by generating a series of mutants that sequentially neutralized three positively charged residues (Lys-49, Lys-53, and Arg-57) within the mouse PLCζ EF-hand domain. Microinjection of the PLCζ EF-hand mutants into mouse eggs enabled their Ca²⁺ oscillation inducing activities to be compared with wild-type PLCζ. Furthermore, the mutant proteins were purified, and the in vitro PIP₂ hydrolysis and binding properties were monitored. Our analysis suggests that PLCζ binds significantly to PIP₂, but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the first EF-hand domain of PLCζ significantly alters in vivo Ca²⁺ oscillation inducing activity and in vitro interaction with PIP₂ without affecting its Ca²⁺ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca²⁺ frequency and the binding ability of different PLCζ mutants to PIP₂. Moreover, a PLCζ mutant with mutations in the cationic residues within the first EF-hand domain and the XY linker region dramatically reduces the binding of PLCζ to PIP₂, leading to complete abolishment of its Ca²⁺ oscillation inducing activity.     

Gelsolin Modulates Phospholipase C Activity In Vivo through Phospholipid Binding

Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to phosphatidylinositol 4,5-bisphosphate (PIP₂) and Ca²⁺ during agonist stimulation. A physiologically relevant rise in Ca²⁺ increases their affinity for PIP₂ and can promote significant interactions with PIP₂ in activated cells. This may impact divergent PIP₂- dependent signaling processes at the level of substrate availability. We found that CapG overexpression enhances PDGF-stimulated phospholipase C_γ (PLC_γ) activity (Sun, H.-q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147–156). In this paper, we examined the ability of gelsolin and CapG to compete with another PLC for PIP₂ in live cells, in semiintact cells, and in vitro. We found that CapG and gelsolin overexpression profoundly inhibited bradykinin-stimulated PLC_β. Inhibition occurred at or after the G protein activation step because overexpression also reduced the response to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells. Conversely, exogenous gelsolin added to permeabilized cells inhibited response in a dose-dependent manner. The washout and addback experiments clearly establish that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLC_β, and only gelsolin domains containing PIP₂-binding sites were effective. Inhibition was mitigated by increasing PIP₂ concentration in a manner consistent with competition between gelsolin and PLC_β for PIP₂. Gelsolin and CapG also had biphasic effects on tyrosine kinase– phosphorylated PLC_γ, although they inhibited PLC_γ less than PLC_β. Our findings indicate that as PIP₂ level and availability change during signaling, cross talk between PIP₂-regulated proteins provides a selective mechanism for positive as well as negative regulation of the signal transduction cascade.     

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.     

Phosphatidylinositol 4,5-Bisphosphate Decreases the Concentration of Ca2+, Phosphatidylserine and Diacylglycerol Required for Protein Kinase C α to Reach Maximum Activity

The C2 domain of PKCα possesses two different binding sites, one for Ca²⁺ and phosphatidylserine and a second one that binds PIP₂ with very high affinity. The enzymatic activity of PKCα was studied by activating it with large unilamellar lipid vesicles, varying the concentration of Ca²⁺ and the contents of dioleylglycerol (DOG), phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphadidylserine (POPS) in these model membranes. The results showed that PIP₂ increased the V_max of PKCα and, when the PIP₂ concentration was 5 mol% of the total lipid in the membrane, the addition of 2 mol% of DOG did not increase the activity. In addition PIP₂ decreases K_0.5 of Ca²⁺ more than 3-fold, that of DOG almost 5-fold and that of POPS by a half. The K_0.5 values of PIP₂ amounted to only 0.11 µM in the presence of DOG and 0.39 in its absence, which is within the expected physiological range for the inner monolayer of a mammalian plasma membrane. As a consequence, PKCα may be expected to operate near its maximum capacity even in the absence of a cell signal producing diacylglycerol. Nevertheless, we have shown that the presence of DOG may also help, since the K_0.5 for PIP₂ notably decreases in its presence. Taken together, these results underline the great importance of PIP₂ in the activation of PKCα and demonstrate that in its presence, the most important cell signal for triggering the activity of this enzyme is the increase in the concentration of cytoplasmic Ca²⁺.     

Divalent cation-dependent formation of electrostatic PIP2 clusters in lipid monolayers

Polyphosphoinositides are among the most highly charged molecules in the cell membrane, and the most common polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate (PIP₂), is involved in many mechanical and biochemical processes in the cell membrane. Divalent cations such as calcium can cause clustering of the polyanionic PIP₂, but the origin and strength of the effective attractions leading to clustering has been unclear. In addition, the question of whether the ion-mediated attractions could be strong enough to alter the mechanical properties of the membrane, to our knowledge, has not been addressed. We study phase separation in mixed monolayers of neutral and highly negatively charged lipids, induced by the addition of divalent positively charged counterions, both experimentally and numerically. We find good agreement between experiments on mixtures of PIP₂ and 1-stearoyl-2-oleoyl phosphatidylcholine and simulations of a simplified model in which only the essential electrostatic interactions are retained. In addition, we find numerically that under certain conditions the effective attractions can rigidify the resulting clusters. Our results support an interpretation of PIP₂ clustering as governed primarily by electrostatic interactions. At physiological pH, the simulations suggest that the effective attractions are strong enough to give nearly pure clusters of PIP₂ even at small overall concentrations of PIP₂.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.     

Cation Penetration through Isolated Leaf Cuticles

The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS⁺ ≥ Rb⁺ > K⁺ > Na⁺ > Li⁺. The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K⁺ and H₂O at pH above 9 were of similar magnitude. At pH 5.5 H₂O penetration was not affected but that of K⁺ was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites.     

Interactive effects of Al, h, and other cations on root elongation considered in terms of cell-surface electrical potential

The rhizotoxicities of Al³⁺ and of La³⁺ to wheat (Triticum aestivum L.) were similarly ameliorated by cations in the following order of effectiveness: H⁺ ≈ C³⁺ > C²⁺ > C¹⁺. Among tested cations of a given charge, ameliorative effectiveness was similar except that Ca²⁺ was slightly more effective than other divalent cations and H⁺ was much more effective than other monovalent cations. H⁺ rhizotoxicity was also ameliorated by cations in the order C³⁺ > C²⁺ > C¹⁺. These results suggest a role for cell-surface electrical potential in the rhizotoxicity of Al³⁺, La³⁺, H⁺, and other toxic cations: negatively charged cell surfaces of the root accumulate the toxic cations, and amelioration is effected by treatments that reduce the negativity of the cell-surface electrical potential by charge screening or cation binding. Membrane-surface activities of free Al³⁺ or La³⁺ computed according to a Gouy-Chapman-Stern model correlated well with growth inhibition, which correlated only poorly with Al³⁺ or La³⁺ activities in the external medium. The similar responses of Al-intoxicated and La-intoxicated roots to ameliorative treatments provide evidence that Al³⁺, rather than AlOH²⁺ or Al(OH)₂⁺, is the principal toxic species of mononuclear Al. Comparisons of the responses of Al-sensitive and Al-tolerant wheats to Al³⁺ and to La³⁺ did not support the hypothesis that varietal sensitivity to Al³⁺ is based upon differences in cell-surface electrical potential.     

Interactions and Diffusion in Fine-Stranded β-lactoglobulin Gels Determined via FRAP and Binding

The effects of electrostatic interactions and obstruction by the microstructure on probe diffusion were determined in positively charged hydrogels. Probe diffusion in fine-stranded gels and solutions of β-lactoglobulin at pH 3.5 was determined using fluorescence recovery after photobleaching (FRAP) and binding, which is widely used in biophysics. The microstructures of the β-lactoglobulin gels were characterized using transmission electron microscopy. The effects of probe size and charge (negatively charged Na₂-fluorescein (376Da) and weakly anionic 70kDa FITC-dextran), probe concentration (50 to 200 ppm), and β-lactoglobulin concentration (9% to 12% w/w) on the diffusion properties and the electrostatic interaction between the negatively charged probes and the positively charged gels or solutions were evaluated. The results show that the diffusion of negatively charged Na₂-fluorescein is strongly influenced by electrostatic interactions in the positively charged β-lactoglobulin systems. A linear relationship between the pseudo-on binding rate constant and the β-lactoglobulin concentration for three different probe concentrations was found. This validates an important assumption of existing biophysical FRAP and binding models, namely that the pseudo-on binding rate constant equals the product of the molecular binding rate constant and the concentration of the free binding sites. Indicators were established to clarify whether FRAP data should be analyzed using a binding-diffusion model or an obstruction-diffusion model.     

Phosphatidylinositol-4,5-Bisphosphate Enhances Anionic Lipid Demixing by the C2 Domain of PKCα

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca²⁺, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP₂) was present in the membrane. When PIP₂ was the labeled phospholipid, in the form of TopFluor-PIP₂, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using ²H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP₂. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP₂. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP₂.     

Fluorescence changes reveal kinetic steps of muscarinic receptor–mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

G protein–coupled receptors initiate signaling cascades. M₁ muscarinic receptor (M₁R) activation couples through Gα_q to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂). Depletion of PIP₂ closes PIP₂-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M₁R activation, M₁R/Gβ interaction, Gα_q/Gβ separation, Gα_q/PLC interaction, and PIP₂ hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M₁R activation (<100 ms) and M₁R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gα_q/Gβ separation and Gα_q/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP₂ hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Gα_q/PLC interaction. Evidently, channel release of PIP₂ and closure are rapid, and the availability of active PLC limits the rate of M current suppression.     

Polyphosphoinositide Phospholipase C in Plasma Membranes of Wheat (Triticum aestivum L.) : Orientation of Active Site and Activation by Ca and Mg

Polyphosphoinositide-specific phospholipase C activity was present in plasma membranes isolated from different tissues of several higher plants. Phospholipase C activities against added phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) were further characterized in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticum aestivum L. cv Drabant) seedlings. In right-side-out (70-80% apoplastic side out) plasma membrane vesicles, the activities were increased 3 to 5 times upon addition of 0.01 to 0.025% (w/v) sodium deoxycholate, whereas in fractions enriched in inside-out (70-80% cytoplasmic side out) vesicles, the activities were only slightly increased by detergent. Furthermore, the activities of inside-out vesicles in the absence of detergent were very close to those of right-side-out vesicles in the presence of optimal detergent concentration. This verifies the general assumption that polyphosphoinositide phospholipase C activity is located at the cytoplasmic surface of the plasma membrane. PIP and PIP₂ phospholipase C was dependent on Ca²⁺ with maximum activity at 10 to 100 μm free Ca²⁺ and half-maximal activation at 0.1 to 1 μm free Ca²⁺. In the presence of 10 μm Ca²⁺, 1 to 2 mm MgCl₂ or MgSO₄ further stimulated the enzyme activity. The other divalent chloride salts tested (1.5 mm Ba²⁺, Co²⁺, Cu²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) inhibited the enzyme activity. The stimulatory effect by Mg²⁺ was observed also when 35 mm NaCl was included. Thus, the PIP and PIP₂ phospholipase C exhibited maximum in vitro activity at physiologically relevant ion concentrations. The plant plasma membrane also possessed a phospholipase C activity against phosphatidylinositol that was 40 times lower than that observed with PIP or PIP₂ as substrate. The phosphatidylinositol phospholipase C activity was dependent on Ca²⁺, with maximum activity at 1 mm CaCl₂, and could not be further stimulated by Mg²⁺.     

Calcium Modulation of Ligand Affinity in the Cyclic GMP–gated Ion Channels of Cone Photoreceptors

To investigate modulation of the activation of cGMP-gated ion channels in cone photoreceptors, we measured currents in membrane patches detached from the outer segments of single cones isolated from striped bass retina. The sensitivity of these channels to activation by cGMP depends on the history of exposure to divalent cations of the membrane''s cytoplasmic surface. In patches maintained in 20 μM Ca⁺⁺ and 100 μM Mg⁺⁺ after excision, the current amplitude dependence on cGMP is well described by a Hill equation with average values of K _1/2, the concentration necessary to activate half the maximal current, of 86 μM and a cooperativity index, n, of 2.57. Exposing the patch to a solution free of divalent cations irreversibly increases the cGMP sensitivity; the average value of K _1/2 shifts to 58.8 μM and n shifts to 1.8. Changes in cGMP sensitivity do not affect other functional parameters of the ion channels, such as the interaction and permeation of mono- and divalent cations. Modulation of cGMP activation depends on the action of an endogenous factor that progressively dissociates from the channel as Ca⁺⁺ concentration is lowered below 1 μM. The activity of the endogenous modulator is not well mimicked by exogenously added calmodulin, although this protein competes with the endogenous modulator for a common binding site. Thus, the modulation of cGMP affinity in cones depends on the activity of an unidentified molecule that may not be calmodulin.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca²⁺ and millimolar concentrations of β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg²⁺ markedly enhances the affinity of the mung bean enzyme for Ca²⁺ but not for β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca²⁺, β-glucoside-activated cotton fiber enzyme, with or without Mg²⁺, is very similar in size to callose isolated from cotton fibers, but is a linear (1→3)-β-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg²⁺in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg²⁺ on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu²⁺ and Cd²⁺. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or β-mercapto-ethanol, while Zn²⁺ or Co²⁺ restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (K_m, 0.55 mM) but that it can hydrolyze this substrate at a high rate (V_max, 30 μmol/min per mg of protein).     

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H: EFFECTS OF DIVALENT METAL IONS*

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca²⁺ and Mg²⁺ and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by ¹⁵N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.     

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (Ca_V) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP₂). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of Ca_V channels by PIP₂. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP₂ sensitivity of Ca_V2.2 and Ca_V1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.