Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum

A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.     

Detection of convergent and parallel evolution at the amino acid sequence level

Adaptive evolution at the molecular level can be studied by detecting convergent and parallel evolution at the amino acid sequence level. For a set of homologous protein sequences, the ancestral amino acids at all interior nodes of the phylogenetic tree of the proteins can be statistically inferred. The amino acid sites that have experienced convergent or parallel changes on independent evolutionary lineages can then be identified by comparing the amino acids at the beginning and end of each lineage. At present, the efficiency of the methods of ancestral sequence inference in identifying convergent and parallel changes is unknown. More seriously, when we identify convergent or parallel changes, it is unclear whether these changes are attributable to random chance. For these reasons, claims of convergent and parallel evolution at the amino acid sequence level have been disputed. We have conducted computer simulations to assess the efficiencies, of the parsimony and Bayesian methods of ancestral sequence inference in identifying convergent and parallel-change sites. Our results showed that the Bayesian method performs better than the parsimony method in identifying parallel changes, and both methods are inefficient in identifying convergent changes. However, the Bayesian method is recommended for estimating the number of convergent-change sites because it gives a conservative estimate. We have developed statistical tests for examining whether the observed numbers of convergent and parallel changes are due to random chance. As an example, we reanalyzed the stomach lysozyme sequences of foregut fermenters and found that parallel evolution is statistically significant, whereas convergent evolution is not well supported.     

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.     

Plasminogen Kanagawa-I, a novel missense mutation, is caused by the amino acid substitution G732R

A new dysplasminogen, plasminogen Kanagawa-I, was identified in a healthy male with no previous thrombotic episodes. His plasma plasminogen (PLG) activity was 51.4% of that of normal pooled plasma (reference interval 70-130%) and the antigen level was 94.2% of that of normal pooled plasma (reference interval 80-150%). Nucleotide sequencing revealed a heterozygous G to A transition in exon 18, which resulted in an amino acid substitution of G732R. Both the proband's father and paternal grandfather were heterozygous for this mutation. Interestingly, the grandfather was found to be a compound heterozygote for plasminogen Kanagawa-I and Tochigi (A601T), so that his plasminogen activity and antigen level was 7.7% and 87.2% of that of normal pooled plasma, respectively. However, he has never been affected by significant thrombosis.     

Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS

The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.     

Thyrotropin receptor cleavage at site 1 does not involve a specific amino acid motif but instead depends on the presence of the unique, 50 amino acid insertion

Thyrotropin (TSH) receptor (TSHR) A and B subunits are formed by intramolecular cleavage of the single chain receptor at two separate sites. The region involved in cleavage at Site 2 has been identified, but previous mutagenesis studies failed to identify Site 1. We now report fortuitous observations on the effect of trypsin on the TSHR that localizes a small region harboring Site 1. Thus, as detected by immunoblotting and by 125I-TSH cross-linking to TSHR expressed on the surface of intact CHO cells, trypsin clipped a small polypeptide fragment bearing a glycan moiety from the C terminus of the A subunit. Based on the TSHR primary structure, this small fragment (1-2 kDa) contains Asn-302. This information, together with estimation of the size of the deglycosylated A subunit relative to a series of C-terminal truncated TSHR ectodomain variants, places cleavage Site 1 in the vicinity of, or closely upstream to, residue 317. Remarkably, mutagenesis of every amino acid residue between residues 298-316 (present study) and 317-362 (previous data) did not prevent cleavage at Site 1. However, cleavage at this site was abrogated by deletion of a 50-amino acid segment (residues 317-366) unique to the TSHR in the glycoprotein hormone receptor family. In summary, these data provide novel insight into TSHR intramolecular cleavage. Cleavage at Site 1 does not depend on a specific amino acid motif and differs from cleavage at Site 2 by involvement of a mechanism requiring the presence of the enigmatic TSHR 50-amino acid "insertion."     

Discordance between malignant hyperthermia susceptibility and RYR1 mutation C1840T in two Scandinavian MH families exhibiting this mutation

The ryanodine receptor 1 (RYR1) mutation C1840T has been reported to segregate with malignant hyperthermia (MH) susceptibility in several families. We have investigated several Scandinavian MH families with respect to five different RYR1 mutations reported to cause predisposition to MH, and we here report on two of the families in which the C1840T mutation was detected. In these two families there was recombination between MH susceptibility and this mutation in one and three individuals, respectively. These findings may suggest that it is necessary to reconsider the specificity of the IVCT and the role of C1840T as a cause of MH susceptibility in some families exhibiting this mutation.     

Effects of serotonin 2 receptor agonists on skeletal muscle preparations in patients with a disposition toward malignant hyperthermia

In pigs genetically susceptible to malignant hyperthermia (MH), it has been shown that serotonin (5-HT2) receptor agonists can induce MH and "psychotic" behaviour. Both can be prevented by 5-HT2 receptor antagonists. Furthermore, free levels of serotonin in plasma increased concomitantly with clinical and laboratory parameters during halothane-induced MH in pigs. In this study the in vitro-effects of the 5-HT2 receptor agonist1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI) were investigated in muscle specimens of MH-susceptible (MHS) and normal (MHN) patients. METHODS. Muscle biopsies were obtained from 37 patients aged 5-69 years (23.6 +/- 5.3 years) with clinical suspicion for MH. The patients were first classified as MHS, MHN or MHE (MH equivocal) by the in vitro contracture test (IVCT) according to the European MH protocol. After MH classification, surplus muscle specimens were subjected to the DOI study. DOI was added to the organ bath in a concentration of 0.02 mmol/l. The in vitro effects on contracture development and muscle twitch were observed for 120 min. RESULTS. Muscle specimens of all patients developed contractures after administration of DOI. However, DOI produced an earlier development of contracture in MHS (17.0 +/- 1.8 min; n = 17) than in MHN (64.7 +/- 5.9 min; n = 15) muscles. In MHS muscles, contractures were more distinct than in MHN muscles; at the end of the experiment, contractures had reached a maximum of 12.5 +/- 0.9 mN in MHS and 5.1 +/- 0.7 mN in MHN muscles. Muscle twitch following DOI administration was reduced significantly in both MHS and MHN muscles. The results of four MHE muscles were comparable with MHS. CONCLUSION. The present study supports the assumption that an altered serotonin system might be involved in the development of MH. In further studies it should investigated whether 5-HT2 receptors of skeletal muscles from MHS subjects are disordered in function or structure. 5-HT2 receptor agonists should be considered as MH-triggering agents.     

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.     

Ten cases of malignant hyperthermia in Norway

Data are presented on ten cases of anaesthesia-induced malignant hyperthermia in Norway. Seven of the patients died, three recovered. The fatal cases were all boys in the age group 11-20 years. This age and sex distribution suggests that puberty with the increase in androgens is a precipitating factor in malignant hyperthermia. One of the victims who survived was a 4 1/2-year-old pseudohermaphrodite girl with the adrenogenital syndrome. The coincidence of malignant hyperthermia in a patient with such a rare syndrome points to the excessive formation of androgens in patients with this syndrome as a predisposing factor. The indications for surgery were traumatic injuries in five cases, congenital abnormalities in three and appendicitis in two cases. These conditions in themselves may cause an increased sensitivity to suxamethonium. One patient received only hexobarbitone, halothane and suxamethonium. After the last drug jaw rigidity and temperature rise to 41.3 degrees C prompted the anaesthetist to end the anaesthetic. The fact that the patient survived proves that suxamethonium induced jaw rigidity is valuable as a warning. The absence of cardiovascular depression after procaine 3.5 g in one patient is ascribed to the correction of acidosis at the time of infusion of this drug. It is suggested that procaine should be withheld until other measures such as cooling, correction of acidosis and steroid therapy have been tried.     

N-system amino acid transport at the blood--CSF barrier

Despite L-glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in L-[14C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na(+)-dependent mechanism. The Na(+)-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na(+)-dependent uptake was inhibited neither by the A-system substrate alpha-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and L-glutamate gamma-hydroxamate, three N-system substrates. Replacement of Na+ with Li+ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The Vmax and Km for glutamine transport by this system were 8.1 +/- 0.3 nmol/mg/min and 3.3 +/- 0.4 mM, respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Absence of association between a mitochondrial DNA mutation at nucleotide position 3243 and schizophrenia in Japanese

PURPOSE: To evaluate the effect that increased numbers of women medical school graduates have had on the composition of orthopedic surgery residencies, and to evaluate trends over time in the likelihood of women medical students to select orthopedic residencies. METHOD: The author analyzed JAMA's "Reports on Graduate and Undergraduate Medical Education" for the years 1977 to 1996, calculating the numbers of women and men in orthopedic surgery and other surgery residencies, and medical school composition. RESULTS: Although there have been modest gains in the number of women in orthopedic surgery training programs in the United States, women continue to choose orthopedics only one-seventh as often as do men. CONCLUSION: Orthopedics remains an unattractive career choice for women medical students compared with their men counterparts. Biases and stereotypes about women and about orthopedic surgery may account for this difference.     

In situ characterization of islets in diabetes with a mitochondrial DNA mutation at nucleotide position 3243

Changes in the pancreas of diabetic patients with the A-to-G mitochondrial DNA (mtDNA) mutation at nucleotide position 3243 base pair (bp) have not previously been described. The clinical phenotypes of diabetes associated with the mtDNA 3243 mutation range from NIDDM to IDDM. We sought the presence of the mutation and studied volume of beta-, alpha-, and delta-cells, mitochondrial enzyme activity, and presence of apoptosis in diabetic pancreases obtained at autopsy. Pancreases were obtained from 16 patients with IDDM, from 18 patients with NIDDM, and from 11 nondiabetic patients. Mitochondrial enzyme activity was determined for cytochrome c oxidase (COX), the subunits of which are partially encoded by mtDNA, and for succinate dehydrogenase (SDH), the subunits of which are solely encoded by nuclear DNA. The volumes of islet beta-, alpha-, and delta-cells were estimated by computerized morphometry. Pancreatic cells were examined for apoptosis by an in situ end-labeling procedure. The mtDNA 3243 mutation was detected in 1 of 16 (6%) pancreases from the IDDM patients; none of the pancreases from 18 NIDDM patients and 11 nondiabetic patients had the mutation. The single patient with the mtDNA 3243 mutation was a 56-year-old woman with IDDM, aged 39 years at diabetes onset, whose mother was diagnosed with NIDDM. The patient had a history of secondary failure of oral hypoglycemic agents and had a marked decrease in the number of beta-cells. The islet beta-cells and non-beta-cells of the patient showed extremely decreased COX enzyme activity. The islet cells in the patient showed a high activity when examined for SDH. Some pancreatic exocrine cells also showed decreased COX activity with high SDH activity. In IDDM, NIDDM, and nondiabetic patients without the mtDNA 3243 mutation, only weak staining for SDH of the islet cells showed. The percentage of heteroplasmy of the mtDNA 3243 mutation in pancreatic micropunched islet specimens was 63 +/- 5% (mean +/- SD) in the islets, 32 +/- 3% in the exocrine pancreas, and 8 +/- 1% in peripheral polymorphonuclear cells. Apoptotic cells were not observed in the IDDM pancreas in the patient with the mtDNA 3243 mutation. The fact that higher levels of mutated mtDNA at 3243 bp were found in affected islets rather than in other tissue suggests that the distribution of the mutant may determine the effect on islet function. A characteristic decrease in the mitochondrial enzyme with COX activity and accelerated SDH activity of the affected islets may provide new insights into the pathogenesis of mitochondrial diabetes.     

Identification of amino acid residues contributing to the pore of a P2X receptor

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.     

Characterization of glucuronic acid conjugates of a novel angiotensin receptor antagonist

Nanogram quantities of glucuronic acid conjugates of GR117289 in rat and dog bile have been analysed by semi-microbore high-performance liquid chromatography (HPLC)/ionspray mass spectrometry with on-line UV diode array detection. The determination of drug metabolites in bile has often proved problematical due to the large number of endogenous components in this biological matrix, in particular the bile acids. Semi-microbore HPLC is useful for concentrating small quantities of material and, in combination with an on-line diode array detector, for distinguishing between drug related and endogenous components. A novel angiotensin II receptor antagonist, GR117289, had proved difficult to analyse by thermospray mass spectrometry because of its thermal lability. The use of the less thermally dependent technique of ionspray mass spectrometry allowed the characterization of nanogram quantities of glucuronic acid metabolites of GR117289 in bile.     

Role of a single amino acid at the amino terminus of the simian virus 5 F2 subunit in syncytium formation

The fusion (F) protein of simian virus 5 (strain W3A) induced extensive cell fusion in BHK cells when expressed alone, while that of strain WR did not. Mutational analysis demonstrated that the fusing activity can be transferred to the WR F protein by a proline residue at position 22 of subunit W3A F2.     

Identification of the amino acid subsets accounting for the ligand binding specificity of a glutamate receptor

In a situation so far unique among neurotransmitter receptors, glutamate receptors share amino acid sequence similarities with the bacterial periplasmic binding proteins (PBPs). On the basis of the primary structure similarity of two bacterial periplasmic proteins (lysine/arginine/ornithine- and phosphate-binding proteins) with the chick cerebellar kainate-binding protein (KBP), a member of the ionotropic glutamate receptor family, we have generated a three-dimensional model structure of the KBP extracellular domain. By an interplay between homology modeling and site-directed mutagenesis, we have investigated the kainate binding properties of 55 different mutants (corresponding to 43 positions) and studied the interactions of some of these mutants with various glutamatergic ligands. As a result, we present here the subsets of amino acids accounting for the binding free energies and specificities of KBP for kainate, glutamate, and CNQX and propose a three-dimensional model, at the microarchitectural level, of the glutamatergic binding domain.     

Cloning of a putative inhibitory amino acid receptor subunit from the parasitic nematode Haemonchus contortus

A cDNA, HGl, encoding an inhibitory amino-acid receptor subunit has been cloned from a mixed egg population of the parasitic nematode Haemonchus contortus. The predicted amino-acid sequence of the subunit shows 24% to 32% homology with other vertebrate and invertebrate GABAA and glycine receptor subunits and has all the expected motifs for a member of the ligand-gated ion channel superfamily. When expressed in Xenopus oocytes HGl gives a small response to 1 mM glycine, but not to 1 mM GABA, glutamate, taurine or L-alanine.     

Detection of a novel Cys628STOP mutation of the myosin VIIA gene in Usher syndrome type Ib

Accessory renal arteries are found frequently--more often on the left side and occurring in as high as 30-35% of cases in some series. These arteries usually enter the upper or lower poles of the kidney. The main clinical significance of such arteries entering the lower pole is that they may obstruct the ureter and lead to hydronephrosis. We report the presence of accessory renal arteries found during routine dissection in an elderly female cadaver. The uniqueness in the variations noted in our cadaver included (1) a dual relationship of the ureters to the accessory renal arteries and (2) both the right and left ovarian arteries originating from their respective accessory arteries. Anomalous renal vessels arise as a result of the complicated development of the kidneys. Similarly, the aberrant origins of both the ovarian arteries observed here could be explained on an embryological basis.