Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.     

Structure/function of the beta-barrel domain of F1-ATPase in the yeast Saccharomyces cerevisiae.

The first 90 amino acids of the alpha- and beta-subunits of mitochondrial F1-ATPase are folded into beta-barrel domains and were postulated to be important for stabilizing the enzyme (Abrahams, J. P., Leslie, A. G., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628). The role of the domains was studied by making chimeric enzymes, replacing the domains from the yeast Saccharomyces cerevisiae enzyme with the corresponding domains from the enzyme of the thermophilic bacterium Bacillus PS3. The enzymes containing the chimeric alpha-, beta-, or alpha- and beta-subunits were not functional. However, gain-of-function mutations were obtained from the strain containing the enzyme with the chimeric PS3/yeast beta-subunit. The gain-of-function mutations were all in codons encoding the beta-barrel domain of the beta-subunit, and the residues appear to map out a region of subunit-subunit interactions. Gain-of-function mutations were also obtained that provided functional expression of the chimeric PS3/yeast alpha- and beta-subunits together. Biochemical analysis of this active chimeric enzyme indicated that it was not significantly more thermostable or labile than the wild type. The results of this study indicate that the beta-barrel domains form critical contacts (distinct from those between the alpha- and beta-subunits) that are important for the assembly of the ATP synthase.     

Transmembrane topology of acetylcholine receptor subunits probed with photoreactive phospholipids

The domains of the acetylcholine receptor subunits that contact the lipid phase were investigated by hydrophobic photolabeling of receptor-rich membrane fragments prepared from Torpedo marmorata and Torpedo californica electric organs. The radioactive arylazido phospholipids used carry a photoreactive group, either at the level of the lipid polar head group (PCI) or at the tip of the aliphatic chain (PCII), and thus probe respectively the "superficial" and "deep" regions of the lipid bilayer. The four subunits of T. marmorata and T. californica acetylcholine receptor reacted with both the PCI and PCII probes and thus are all exposed to the lipid phase. Ligands known to stabilize different conformations of the acetylcholine receptor (nicotinic agonists, snake alpha-toxin, and noncompetitive blockers) did not cause any significant change in the labeling pattern. The acetylcholine receptor associated 43 000-dalton v1 protein did not react with any of the probes. A striking difference in labeling between T. marmorata and T. californica acetylcholine receptors occurred at the level of the alpha-subunit when the superficial PCI probe was used. An approximately 5-fold higher labeling of the alpha-subunit as compared to the beta-, gamma-, and delta-subunits was observed by using receptor-rich membranes from T. marmorata but not from T. californica. The same difference persisted after purification of the labeled receptors from the two species and was restricted to an 8000-dalton C-terminal tryptic peptide. The only mutation observed in this region of the complete alpha-subunit sequence of the two species is the substitution of cysteine-424 in T. marmorata by serine-424 in T. californica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae.

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.     

Assembly of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase with its pre-existing beta-subunit in Xenopus oocytes

The alpha- and beta-subunits of Torpedo californica Na+/K(+)-ATPase were expressed in turn in single oocytes by alternately microinjecting the specific mRNAs for the alpha- and beta-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing beta-subunit, which had been synthesized by injecting mRNA for the beta-subunit, could assemble with the alpha-subunit expressed later in the single oocytes and the resulting alpha beta complex acquired both ouabain-binding and Na+/K(+)-ATPase activities. On the other hand, formation of the alpha beta complex was not detected when the alpha-subunit was expressed first, followed by the beta-subunit. These data suggest that the beta-subunit acts as a receptor or a stabilizer for the alpha-subunit upon the biogenesis of Na+/K(+)-ATPase.     

Phosphorylation of phosphatidylinositol associated with the nicotinic acetylcholine receptor of Torpedo californica

When isolated, detergent solubilized and affinity chromatographically purified nicotinic acetylcholine receptor of Torpedo californica electric organ is incubated with [gamma-32P]ATP/Mg2+, phosphatidylinositol 4-phosphate (PIP) is formed from receptor associated phosphatidylinositol (PI). This receptor associated endogenous kinase activity is enhanced by orthovanadate and, remarkably, also by acetylcholine. Exogenously added PI-kinase only increases the phosphorylation rate if vanadate is present. PIP as the main phosphorylation product (up to 95%) remains bound to the beta-, gamma- and delta-subunits of the receptor and to the receptor associated v-protein. The alpha-subunits do not carry 32p phosphate; no phosphatidylinositol 4,5-bisphosphate formation has been observed. Concomitant to lipid phosphorylation tyrosine and serine residues are phosphorylated (5% of total incorporated 32P phosphate).     

Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle.

The isolation and characterization of five clones carrying sequences of the alpha-, beta-, gamma-, delta- and epsilon-subunit precursors of the rat muscle acetylcholine receptor (AChR) are described. The deduced amino acid sequences indicate that these polypeptides contain 457-519 amino acids and reveal the structural characteristics common to subunits of ligand-gated ion channels. The pattern of subunit-specific mRNA levels in rat muscle shows characteristic changes during development and following denervation, suggesting that innervation of muscle reduces the expression of the alpha-, beta- and delta-subunit mRNAs, suppresses the expression of the gamma-subunit mRNA, and induces expression of epsilon-subunit mRNA. Subunit-specific cRNAs generated in vitro were injected into Xenopus laevis oocytes, resulting in the assembly of two functionally different AChR channel subtypes. The AChR gamma, composed of the alpha-, beta-, gamma- and delta-subunits, has functional properties similar to those of the native AChRs in fetal muscle. The AChR epsilon, composed of alpha-, beta-, delta- and epsilon-subunits, corresponds to the end-plate channel of the adult muscle. Thus in rat skeletal muscle the motor nerve regulates the expression of two functionally different AChR subtypes with different molecular composition by the differential expression of subunit-specific mRNAs.     

Transport of the yeast ATP synthase beta-subunit into mitochondria. Effects of amino acid substitutions on targeting.

We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence. A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria. A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease. Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA. The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins. All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate. The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant. More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.     

Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.     

Structure of the extra-membranous domain of the beta-subunit of (Na,K)-ATPase revealed by the sequences of its tryptic peptides

Membrane bound dog kidney (Na,K)-ATPase was digested with trypsin. The peptides that were recovered in the supernatant were purified and sequenced. By comparing these results with the sequence of alpha- and human beta-subunits, the location of each of the peptides could be allotted. Both accessibility to trypsin and the facility of release into the water phase indicated that these peptides were derived from the exposed surface of the intact enzyme. The sequence, GXGXXG, reported in the Torpedo californica beta-subunit [(1986) FEBS Lett. 196, 315-319] was likely a mere coincidence with the sequence of the dinucleotide-binding site, since the last glycine was replaced by proline in the sequence of the dog beta-subunit. A disulfide bridge was found within a peptide derived from the beta-subunit. A possible model for the beta-subunit structure is proposed.     

A possible role of the beta-subunit of (Na,K)-ATPase in facilitating correct assembly of the alpha-subunit into the membrane

mRNAs from the alpha- and beta-subunits (mRNA alpha and mRNA beta, respectively) of Torpedo californica (Na,K)-ATPase were injected into Xenopus laevis oocytes either separately or in combination, and the properties of the two subunits synthesized were studied. The alpha-subunit synthesized in oocytes injected with mRNA alpha alone was recovered in both the membrane and cytosol fractions and was susceptible to tryptic attack. When mRNA beta was coinjected with mRNA alpha, almost all the alpha-subunit was found in the membrane fraction and was resistant to trypsin. In all cases, essentially all of the beta-subunit was recovered in the membrane fraction and was resistant to trypsin. As the amount of mRNA beta coinjected increased, the amounts of both the alpha- and beta-subunits as well as (Na,K)-ATPase activity of the membrane fraction increased. These results suggest that the beta-subunit facilitates the correct assembly of the alpha-subunit into the membrane probably by forming a stable complex with the nascent alpha-subunit.     

Factors determining the subunit composition of tropomyosin in mammalian skeletal muscle.

Adult rat fast-twitch skeletal muscle such as extensor digitorum longus contains alpha- and beta-tropomyosin subunits, as is the case in the corresponding muscles of rabbit. Adult rat soleus muscle contains beta-, gamma- and delta-tropomyosins, but no significant amounts of alpha-tropomyosin. Evidence for the presence of phosphorylated forms of at least three of the four tropomyosin subunit isoforms was obtained, particularly in developing muscle. Immediately after birth alpha- and beta-tropomyosins were the major components of skeletal muscle, in both fast-twitch and slow-twitch muscles. Differentiation into slow-twitch skeletal muscles was accompanied by a fall in the amount of alpha-tropomyosin subunit and its replacement with gamma- and delta-subunits. After denervation and during regeneration after injury, the tropomyosin composition of slow-twitch skeletal muscle changed to that associated with fast-twitch muscle. Thyroidectomy slowed down the changes in tropomyosin composition resulting from the denervation of soleus muscle. The results suggest that the 'ground state' of tropomyosin-gene expression in the skeletal muscle gives rise to alpha- and beta-tropomyosin subunits. Innervation by a 'slow-twitch' nerve is essential for the expression of the genes controlling gamma- and delta-subunits. There appears to be reciprocal relationship between expression of the gene controlling the synthesis of alpha-tropomyosin and those controlling the synthesis of gamma- and delta-tropomyosin subunits.     

Pig kidney Na+,K+-ATPase. Primary structure and spatial organization

cDNAs complementary to pig kidney mRNAs coding for alpha- and beta-subunits of Na+,K+-ATPase were cloned and sequenced. Selective tryptic hydrolysis of the alpha-subunit within the membrane-bound enzyme and tryptic hydrolysis of the immobilized isolated beta-subunit were also performed. The mature alpha- and beta-subunits contain 1016 and 302 amino acid residues, respectively. Structural data on the peptides from extramembrane regions of the alpha-subunit and on glycopeptides of the beta-subunit underlie a model for the transmembrane arrangement of Na+,K+-ATPase polypeptide chains.     

Down-regulation of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition

Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the alpha- and/or beta-subunit or arises from a physical dissociation of the alpha-subunit from the beta-subunit. The beta-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using alpha- and beta-subunit-specific antibodies, detected bands of 110-125 and 36 kDa, respectively. Protein expression levels of the alpha-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, alpha-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased beta-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the alpha- and beta-subunits in samples taken before or after the onset of labor. We conclude that during labor, the alpha-subunit is not physically uncoupled from the beta-subunit, but a decline occurs in the level of beta-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced beta-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.     

Asymmetry of the rat acetylcholine receptor subunits in the narrow region of the pore.

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the alpha-subunit revealed that a threonine residue (alpha T264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the beta-, gamma-, and delta-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the alpha-, beta-, or gamma-subunits, but it is smaller if the valine is located in the delta-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of alpha-, gamma- and delta-subunits, where the delta-subunit serine is a main determinant of the channel conductance.     

Expression of Torpedo nicotinic acetylcholine receptor subunits in yeast is enhanced by use of yeast signal sequences

We have produced the four subunits of the nicotinic acetylcholine receptor of Torpedo californica, an integral membrane protein, in the yeast Saccharomyces cerevisiae. Two of the subunits (alpha and delta) were readily produced from their cDNAs after simply subcloning them into a yeast shuttle vector adjacent to a yeast promoter. The other two protein subunits (beta and gamma) were not produced by this strategy, although the amounts of mRNA produced from these expression constructs are similar to those for alpha and delta. Replacing the DNA coding for the normal N-terminal signal sequences for the beta and gamma subunits with DNA coding for the signal sequence of yeast invertase results in successful protein synthesis. The yeast signal sequence allows these subunits to be translocated across the membrane of the endoplasmic reticulum and to be glycosylated. The appropriate final size of the subunit proteins suggests that the yeast signal sequence has been properly cleaved after translocation.     

Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS-1: a hetero-oligomeric chaperonin with variable subunit composition

To study the difference in expression of the chaperonin alpha- and beta-subunits in Thermococcus strain KS-1 (T. KS-1), we measured their intracellular contents at various growth temperatures using subunit-specific antibodies. The beta-subunit was significantly more abundant with increasing temperature (maximum at 93 degrees C), whereas the alpha-subunit was not. Native PAGE with Western blot analysis indicated that the natural chaperonins in the crude extracts of T. KS-1 cells grown between 65 degrees C and 95 degrees C migrate as single bands with different mobility. The recombinant alpha- and beta-subunit homo-oligomers migrated differently from each other and from natural chaperonins. Immunoprecipitation also showed that the natural chaperonin was the hetero-oligomer. These results indicate that chaperonin in T. KS-1 formed a hetero-oligomer with variable subunit composition, and that the beta-subunit may be adapted to a higher temperature than the alpha-subunit. T. KS-1 probably changes its chaperonin subunit composition to acclimatize to the ambient temperature.     

Molecular weight of the acetylcholine receptors of electric organs and the effect of Triton X-100.

Studies are reported on the influence of Triton X-100 on the molecular weight and functional properties of the acetylcholine receptor. Results are presented principally for receptors purified from Torpedo californica and Torpedo marmorata with a limited number of observations on the receptor from Electrophorus electricus. In equilibrium dialysis measurements Trito, X-100 greatly reduced acetylcholine binding to the high affinity sites of the receptor from T. californica, but had only a small effect on the sites of lower affinity. Sedimentation equilibrium experiments on receptor in the absence of added Triton X-100 revealed average apparent molecular weight values of 510,000 for receptor from T. californica and 665,000 for T. marmorata. Under those conditions 0.113 mg of residual Triton X-100 were found per mg of protein as determined by using 3H-labeled Triton X-100. The sedimentation data indicated the presence of more than one molecular species, involving a unit with an apparent molecular weight of 330,000 and higher aggregates. Upon addition of Triton X-100, the higher aggregates were reduced, and above 0.1 percent Triton X-100 the 330,000 unit was the principal component present for receptor from all three species examined. Various structural models are considered in the light of this value, the polypeptide size from Na dodecyl sulfate-gel electrophoresis, and the protomer size determined by the molecular weight of an acetylcholine binding site.     

Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.