Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles

Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides.     

Monomers and oligomers of the M2 muscarinic cholinergic receptor purified from Sf9 cells

G protein-coupled receptors are known to form oligomers. To probe the nature of such aggregates, as well as the role and prevalence of monomers, epitope-tagged forms of the M(2) muscarinic receptor have been isolated as oligomers and monomers from Sf9 cells. Membranes from cells coexpressing the c-Myc- and FLAG-tagged receptor were solubilized in digitonin-cholate, and the receptor was purified by successive passage through DEAE-Sepharose, the affinity resin 3-(2'-aminobenzhydryloxy)tropane (ABT)-Sepharose, and hydroxyapatite. Coimmunoprecipitation of the two epitopes indicated the presence of oligomers at each stage of the purification up to but not including the fraction eluted specifically from ABT-Sepharose. The affinity-purified receptor therefore appeared to be monomeric. The failure to detect coimmunoprecipitation was not due to an ineffective antibody, nor did the conditions of purification appear to promote disaggregation. Receptor at all stages of purification bound N-[(3)H]methylscopolamine and [(3)H]quinuclidinylbenzilate with high affinity, but the capacity of receptors that were not retained on ABT-Sepharose was only 4% of that expected from densitometry of western blots probed with an anti-M(2) antibody. Similarly low activity was found with oligomers isolated by successive passage of coexpressed receptor on anti-c-Myc and anti-FLAG immunoaffinity columns. M(2) muscarinic receptors therefore appear to coexist as active monomers and largely or wholly inactive oligomers in solubilized extracts of Sf9 cells. A different pattern emerged when coinfected cells were treated with quinuclidinylbenzilate prior to solubilization, in that ABT-purified receptors from those cells exhibited coimmunoprecipitation. Treatment with the antagonist therefore led to oligomers in which at least some of the constituent sites were active and were retained by ABT-Sepharose.     

Oligomeric potential of the M2 muscarinic cholinergic receptor

G protein-coupled receptors are known to exist as oligomers. Although such aggregates often are referred to as dimers, there is little direct evidence regarding their oligomeric size. In the present investigation, c-Myc-, FLAG-, and influenza hemagglutinin (HA)-tagged forms of the M2 muscarinic receptor have been coexpressed in Sf9 cells to probe for aggregates larger than a dimer. Immunochromatography, immunoprecipitation, and immunoblotting were carried out with various combinations of antibodies directed against the different epitopes to demonstrate that all three tagged forms of the receptor can be immunopurified within a single complex. Extracts of the M2 muscarinic receptor from Sf9 cells therefore contain aggregates that are at least trimeric, and the levels detected point to the existence of larger complexes. The data also suggest that the oligomers coexist with a sizeable population of monomers.     

Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by phosphatidylserine

The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation. Homogeneous tissue factor, a glycoprotein purified from bovine brain, was reconstituted into phospholipid vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (40% phosphatidylserine) with octyl glucoside. The vesicles were characterized with respect to size and tissue factor content and orientation. Employing data from protease digestion, we deduced that tissue factor is randomly oriented; thus, its effective concentration in these vesicles was half its total concentration. In all binding experiments, 1 mol of enzyme was bound per mole of available activator at saturation. This stoichiometry was not affected by the form of the enzyme employed or the phospholipid composition of the vesicles. With tissue factor incorporated into phosphatidylcholine vesicles, the Kd was 13.2 +/- 0.72 nM for factor VII and 4.54 +/- 1.37 nM for factor VIIa. Thus, the one-chain zymogen binds to the activator with only slightly less affinity than the more active two-chain enzyme. Active-site modification of factor VII and factor VIIa with diisopropyl fluorophosphate resulted in tighter binding of the derivatized molecules. Inclusion of phosphatidylserine in the vesicles altered the binding both quantitatively and qualitatively. With increasing acidic phospholipid, the concentration of enzyme required to occupy half the activator sites was decreased. In addition, positive cooperativity was observed, the degree of which depended on the vesicle charge and the form of the enzyme. An explicit two-site cooperative binding model is presented which fits these complex data. In this model, tissue factor is at least a dimer with two interacting enzyme binding sites.     

Investigations into the physiological role of muscarinic M2 and M4 muscarinic and M4 receptor subtypes using receptor knockout mice

Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.     

Magnetic resonance study of 13C-enriched glycophorin A reconstituted into phospholipid vesicles

$\text{[S-[}{}^{13}\text{C}\text{]}\text{methylmethionine-8 and -81]glycophorin}$ A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethyionine}\text{-81}$ residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethionine}\text{-8}$ residue.     

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.     

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.     

Functional interaction of purified muscarinic receptors with purified inhibitory guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

The GTP binding regulatory protein (Ni involved in adenylate cyclase inhibition was purified from rat brain and reconstituted, together with muscarinic cholinergic receptors purified from porcine brain, into phospholipid vesicles. Guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding and GTP hydrolyzing activities of reconstituted Ni were stimulated by the addition of a muscarinic agonist, carbachol. The effect of carbachol was to increase the Vmax values of these activities, but the Km values were also increased slightly in most cases. Carbachol bound to vesicles with the same order of magnitude of Km as that for stimulation of GTPase. The affinity of this binding was reduced by GTP gamma S, indicating that the high-affinity receptor-Ni complex was formed in a GTP-dependent manner in reconstituted vesicles. Incubation of Ni with NAD and islet-activating protein (IAP), pertussis toxin, caused ADP-ribosylation of the alpha-subunit of Ni. The criteria for the receptor-Ni interaction, i.e. carbachol stimulation of the activities of Ni and the GTP gamma S effect on carbachol binding, were no longer observed, when this IAP-treated Ni, instead of the nontreated Ni, was reconstituted into vesicles, though there was no difference between IAP-treated and nontreated Ni in their basal activities observable without carbachol. No, the protein with a character very similar to Ni in rat brain, was also coupled to muscarinic receptors when they were reconstituted into vesicles under the same conditions. Thus, GTP-binding proteins serving as the substrate of IAP-catalyzed ADP-ribosylation are capable of interaction functionally with muscarinic receptors in phospholipid vesicles.     

Incorporation of the purified human placental insulin receptor into phospholipid vesicles

Purified human placental insulin receptors were incorporated into small unilamellar phospholipid vesicles by the addition of n-octyl beta-glucopyranoside solubilized phospholipids, followed by removal of the detergent on a Sephadex G-50 gel filtration column and extensive dialysis. The vesicles have an average diameter of 142 +/- 24 nm by Sephacryl S-1000 gel filtration chromatography and 119 +/- 20 nm by transmission electron microscopy. These vesicles are impermeant to small molecules as indicated by their ability to retain [gamma-32P]ATP, which could be released by the addition of 0.05% Triton X-100. Detergent permeabilization or freeze-thawing of the insulin receptor containing vesicles in the presence of 125I-insulin indicated that approximately 75% of the insulin binding sites were oriented right side out (extravesicularly). Sucrose gradient centrifugation of insulin receptors incorporated at various protein to phospholipid mole ratios demonstrated that the insulin receptors were inserted into the phospholipid bilayer structure in a concentration-dependent manner. Addition of [gamma-32P]ATP to the insulin receptor containing vesicles was relatively ineffective in promoting the autophosphorylation of the beta subunit in the absence or presence of insulin. Permeabilization of the vesicles with low detergent concentrations, however, stimulated the beta-subunit autophosphorylation approximately 2-fold in the absence and 10-fold in the presence of insulin. Insulin-stimulated beta-subunit autophosphorylation was also observed under conditions such that 94% of those vesicles containing insulin receptors had a single receptor per vesicle, suggesting that the initial beta-subunit autophosphorylating activity is intramolecular. Phospho amino acid analysis of the vesicle-incorporated insulin receptors demonstrated that the basal and insulin-stimulated beta-subunit autophosphorylation occurs exclusively on tyrosine residues. It is concluded that when purified insulin receptors are incorporated into a phospholipid bilayer, they insert into the vesicles primarily in the same orientation as occurs in the plasma membrane of intact cells and retain insulin binding as well as insulin-stimulated beta-subunit autophosphorylating activities.     

Ultrastructure of reconstituted membranes containing the muscarinic receptor

In this paper the demonstration is made that membrane vesicles (liposomes) containing the muscarinic receptor can be formed by polyethylene glycol (PEG) precipitation of detergent extracts of bovine atrial membranes. The incorporation of the muscarinic receptor in these vesicles may be related to the restoration of the heterogeneity and nucleotide modulation of muscarinic agonist binding by PEG precipitation of atrial detergent extracts, previously reported. Vesicles are also formed when detergent solubilized asolectin lipids, alone or in combination with membrane detergent extracts, are precipitated by PEG. The structure of the vesicles seems depend on their lipid and protein composition and the procedure employed for the removal of the dispersing medium. These results indicate that PEG precipitation could be used for the reconstitution of the muscarinic receptor into the liposomes of exogenous lipids.Special Issue dedicated to Prof. Eduardo De Robertis.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

Role of internalization of M2 muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+ current in heart

In the heart, ACh activates the ACh-activated K(+) current (I(K,ACh)) via the M(2) muscarinic receptor. The relationship between desensitization of I(K,ACh) and internalization of the M(2) receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, I(K,ACh) declined by approximately 62% with time constants of 1.5 and 26.9 min, whereas approximately 83% of the M(2) receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with beta-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and beta-arrestin 2 significantly increased I(K,ACh) desensitization and M(2) receptor internalization during a 3-min application of agonist. Internalized M(2) receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and beta-arrestin 2-dependent internalization of the M(2) receptor into clathrin-coated vesicles could play a major role in I(K,ACh) desensitization.     

ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin

The coupling factor ATPase complex extracted by Triton X-100 from the photosynthetic bacterium Rhodospirillum rubrum could be incorporated into phospholipid vesicles after removal of the Triton. Vesicles reconstituted with this F₀ · F₁-type ATPase together with bacteriorhodopsin were found to catalyze, in the light, net ATP synthesis which was inhibited by the energy transfer inhibitors oligomycin and N,N-dicyclohexylcarbodiimide as well as by uncouplers. In vesicles reconstituted with the crude ATPase up to 50% of the observed rate of phosphorylation was independent on light and bacteriorhodopsin and insensitive to the above-listed inhibitors. This dark activity was, however, completely blocked by the adenylate kinase inhibitor, p¹,p⁵-di(adenosine-5′)pentaphosphate, which did not affect at all the net light-dependent phosphorylation nor the ATP-³²P_i exchange reaction. Vesicles reconstituted with the purified ATPase catalyzed only the light- and bacteriorhodopsin-dependent diadenosine pentaphosphate-insensitive phosphorylation. The rate of this photophosphorylation was found to be proportional to the amount of ATPase and bacteriorhodopsin, and linear for at least 20 min of illumination. These results indicate that the purified ATPase contains the complete assembly of subunits required to transduce electrochemical gradient energy into chemical energy.     

Amplification of the rat M2 muscarinic receptor gene by the polymerase chain reaction: functional expression of the M2 muscarinic receptor

A selective amplification of the coding sequence of the rat M2 muscarinic receptor gene was achieved by the polymerase chain reaction. The error rate of this amplification system under conditions specified was 1 nucleotide substitution in 841 base pairs. In vitro expression of this gene in murine fibroblasts (B82) via the eukaryotic expression vector, pH beta APr-1-neo, resulted in high level expression of specific [3H] (-)MQNB binding in transfected B82 cell lines. One of these clones, M2LKB2-2, showed a stable expression of [3H] (-)MQNB binding with a Kd value of 265 pM and a Bmax value of 411 +/- 50 fmol/10(6) cells. Cardiac selective muscarinic antagonists such as himbacine and AF-DX 116 show high affinities for this binding site in the M2LKB2-2 cells. The rank order of potency of several antagonists in inhibiting [3H] (-)MQNB binding in these cells conformed to the characteristics of an M2 type muscarinic receptor. Carbachol showed a single affinity state for the receptors in the M2LKB2-2 cells with a Ki value of 2.0 microM. This receptor appeared to be inversely coupled to adenylate cyclase via a pertussis toxin sensitive G-protein. Carbachol also had a slight stimulatory effect on the hydrolysis of inositol lipids. The polymerase chain reaction proves highly effective in cloning genes from genomic material, as demonstrated by the first in vitro functional expression of the rat M2 type muscarinic receptor.     

Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles

Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis. We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution. Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles. The reconstituted receptors still bound radioligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors.     

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+ pump by phospholamban in reconstituted phospholipid vesicles

Phospholamban is the regulator of the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum (SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban, and phosphorylation may relieve this inhibition. Fast-twitch skeletal muscle SR does not contain phospholamban, and it is not known whether the Ca(2+)-ATPase isoform from this muscle may be also subject to regulation by phospholamban in a similar manner as the cardiac isoform. To determine this we reconstituted the skeletal isoform of the SR Ca(2+)-ATPase with phospholamban in phosphatidylcholine proteoliposomes. Inclusion of phospholamban was associated with significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0, and phosphorylation of phospholamban by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects on the Ca2+ pump. Similar effects of phospholamban were also observed using phosphatidylcholine:phosphatidylserine proteoliposomes, in which the Ca2+ pump was activated by the negatively charged phospholipids (24). Regulation of the Ca(2+)-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by cross-linking experiments, using a synthetic peptide that corresponded to amino acids 1-25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca(2+)-ATPase may be also regulated by phospholamban, although this regulator is not expressed in fast-twitch skeletal muscles.     

Fusion of phospholipid vesicles reconstituted with cytochromec oxidase and mitochondrial hydrophobic protein

Summary Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes and Ca⁺⁺ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca⁺⁺ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 Å; fusion yielded vesicles with diameters in excess of 1000 Å.     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.