M1 Region Research Articles

Image-Subtraction Photometry of the Globular Cluster M3: Identification of New Double-Mode RR Lyrae Stars

We have applied the image subtraction method of Alard and Lupton to the extensive M3 data set previously analyzed by Corwin and Carney using DAOPHOT and ALLSTAR. This new analysis has produced light curves and periods for 15 variables not found in the previous study but already known to be variables, and it has also resulted in improved periods for several other variables. The additional variables recovered with the image subtraction analysis are in the very central region of M3, where crowding is severe and the photometry was not of sufficient quality that it could be put on the standard system. The present study brings to 222 the total number of RR Lyrae variables in Corwin and Carney's M3 data set for which light curves and periods are available. Among them we have identified three new candidate double-mode pulsating variables (V13, V200, and V251), reported here for the first time. This brings to eight the total number of double-mode RR Lyrae (RRd's) identified in M3. Of the newly discovered RRd's V13 is unusual in that it has the fundamental as the dominant pulsation mode. M3 is unique among the globular clusters in having RRd variables with a dominant fundamental mode. Two of the new candidate RRd's (V13 and V200) have period ratios as low as 0.738–0.739. They lie well separated from all previously known double-mode variable stars in the Petersen diagram in positions implying a large spread in mass and/or, less likely, in heavy element mass fraction, among the M3 horizontal-branch (HB) stars. We explore mass transfer and helium enhancement as possible explanations for the apparent spread in HB masses. We also note that the masses derived from the double-mode analyses now favor little mass loss on the red giant branch. We find that V200 has changed its dominant pulsation mode from fundamental to first overtone, while V251 has changed its dominant mode from first overtone to fundamental in the interval 1992 to 1993. Together with M3-V166 this is the first time that double-mode variables are observed to switch their dominant pulsation modes while remaining RRd's. The phenomenon is found to occur in a 1 yr time span, thus suggesting that these stars are undergoing a rapid evolutionary phase and that both redward and blueward evolution may take place among the HB stars in the Oosterhoff type I cluster M3. The unusual behavior of the M3 RRd's is discussed in detail and compared with that of the double-mode RR Lyrae identified so far in globular clusters and in the field of our and other Local Group galaxies. We find a lack of correlation between the presence of RRd variables and any of the cluster structural parameters.

MAEBL Plasmodium falciparum protein peptides bind specifically to erythrocytes and inhibit in vitro merozoite invasion

MAEBL is an erythrocyte binding protein located in the rhoptries and on the surface of mature merozoites, being expressed at the beginning of schizogony. The structure of MAEBL originally isolated from rodent malaria parasites suggested a molecule likely to be involved in invasion. We thus became interested in identifying possible MAEBL functional regions. Synthetic peptides spanning the MAEBL sequence were tested in erythrocyte binding assays to identify such possible MAEBL functional regions. Nine high activity binding peptides (HABPs) were identified: two were found in the M1 domain, one was found between the M1 and M2 regions, five in the erythrocyte binding domain (M2), and one in the protein’s repeat region. The results showed that peptide binding was saturable; some HABPs inhibited in vitro merozoite invasion and specifically bound to a 33 kDa protein on red blood cell membrane. HABPs’ possible function in merozoite invasion of erythrocytes is also discussed.

Illusory movements of the paralyzed limb restore motor cortex activity

In humans, limb amputation or brachial plexus avulsion (BPA) often results in phantom pain sensation. Actively observing movements made by a substitute of the injured limb can reduce phantom pain (V.S. Ramachandran and D. Rogers-Ramachandran, 1996, Proc. R. Soc. London B Biol. Sci. 263, 377–386). The neural basis of phantom limb sensation and its amelioration remains unclear. Here, we studied the effects of visuomotor training on motor cortex (M1) activity in three patients with BPA. Functional magnetic resonance imaging scans were obtained before and after an 8-week training program during which patients learned to match voluntary “movements” of the phantom limb with prerecorded movements of a virtual hand. Before training, phantom limb movements activated the contralateral premotor cortex. After training, two subjects showed increased activity in the contralateral primary motor area. This change was paralleled by a significant reduction in phantom pain. The third subject showed no increase in motor cortex activity and no improvement in phantom pain. We suggest that successful visuomotor training restores a coherent body image in the M1 region and, as a result, directly affects the experience of phantom pain sensation. Artificial visual feedback on the movements of the phantom limb may thus “fool” the brain and reestablish the original hand/arm cortical representation.

Functional Consequences of Alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal Part of Transmembrane Helix M3 of the Na+,K+-ATPase

Mutations Ile279 --> Ala, Ile283 --> Ala, Glu284 --> Ala, His285 --> Ala, His285 --> Lys, His285 --> Glu, Phe286 --> Ala, and His288 --> Ala in transmembrane helix M3 of the Na+,K(+)-ATPase were studied. Except for His285 --> Ala, these mutations were compatible with cell viability, permitting analysis of their effects on the overall and partial reactions of the Na+,K(+)-transport cycle. In Ile279 --> Ala and Ile283 --> Ala, the E1 form accumulated, whereas in His285 --> Lys and His285 --> Glu, E1P accumulated. Phe286 --> Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E2 and E2P, respectively, and showed a unique enhancement of the E1P --> E2P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E1-E2 and E1P-E2P conformational changes. Because the E1-E2 and E1P-E2P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His285 furthermore increased the Na(+)-activated ATPase activity in the absence of K+ ("Na(+)-ATPase activity"). Ile279 --> Ala, Ile283 --> Ala, and His288 --> Ala showed reduced Na+ affinity of the E1 form. The rate of Na(+)-activated phosphorylation from ATP was reduced in Ile279 --> Ala and Ile283 --> Ala, and these mutants showed evidence similar to Glu329 --> Gln of destabilization of the Na(+)-occluded state.

Extracellular vestibule determinants of Ca2+ influx in Ca2+-permeable AMPA receptor channels.

At certain synapses in the brain, Ca2+-permeable AMPA receptor (AMPAR) channels represent an important pathway for synaptically controlled Ca2+ entry. However, the molecular determinants of this Ca2+ influx are poorly defined. In NMDA receptor (NMDAR) channels, where the influx is much greater, the extracellular vestibule, specifically the M3 segment and regions C-terminal to it in the NR1 subunit, contains elements critical to their high Ca2+ influx under physiological conditions. We therefore investigated the contribution of homologous positions in AMPAR as well as kainate receptor (KAR) subunits to the process of Ca2+ influx. Substitutions of a conserved asparagine (N) in M3 of AMPAR GluR-B(Q) channels strongly attenuated Ca2+ permeability measured using reversal potentials under biionic conditions and fractional Ca2+ currents recorded under physiological conditions. Hence, as in NMDAR channels, the conserved N makes a significant contribution to Ca2+ influx in AMPAR channels. In addition, C-terminal to M3, substitutions of negatively (glutamate, E) or positively (arginine, R) charged residues also altered Ca2+ influx. However, in contrast to charged residues occupying homologous positions in NMDAR channels, these effects were about equal and opposite suggesting that this ER in AMPARs does not contribute significantly to the mechanism of Ca2+ influx. Opposite charge substitutions of two negative residues C-terminal to M3 in KAR GluR-6(Q) subunits had no effect on Ca2+ permeability. We conclude that the different contribution of residues C-terminal to M3 to Ca2+ permeation in NMDAR and non-NMDAR channels reflects a different positioning of these residues relative to the tip of the M2 loop.

Open-State Disulfide Crosslinking between Mycobacterium tuberculosis Mechanosensitive Channel Subunits

The mechanosensitive channel of large conductance from Mycobacterium tuberculosis (Tb-MscL) was subjected to cysteine-scanning mutagenesis at several residues in the M1 region. The V15C channel displayed disulfide crosslinking in air, but not in the presence of 100 mM β-mercaptoethanol. In single-channel experiments, the V15C channel was more sensitive to tension than was wild-type Tb-MscL. In air, Tb-MscL V15C occasionally displayed signature-events: at constant tension, there was first a sojourn in the highest conductance open state, then a series of transitions to substates. During a signature-event, these transitions do not appear to be reversible. Some sojourns in the lower conductance states lasted for ≥100 s. These signature-events were abolished by 100 mM β-mercaptoethanol and did not occur in a cysteineless gain-of-function mutant, suggesting that the signature-events represent disulfide crosslinking between channel subunits. We conclude that the crosslinking occurs during an open state during asymmetric sojourns that bring the α-carbons of adjacent 15C side chains within 3.6–6.8 Å. Such asymmetric structures must be considered in models of TB-MscL gating.

Increased Sensitivity to Agonist-Induced Seizures, Straub Tail, and Hippocampal Theta Rhythm in Knock-In Mice Carrying Hypersensitive α4 Nicotinic Receptors

We studied a strain of exon replacement mice ("L9'S knock-in") whose alpha4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9' position (Labarca et al., 2001); this mutation renders alpha4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9'S than in wild-type (WT) littermates. At these concentrations, L9'S but not WT showed increases in EEG amplitude and theta rhythm. L9'S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA(A) receptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9'S mice (0.1 mg/kg). The L9'S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9'S and WT mice in several brain regions. [(125)I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at approximately 25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. (86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9'S mice. These data support the possible involvement of gain-of-function alpha4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy.

First and second transmembrane segments of alpha3, alpha4, beta2, and beta4 nicotinic acetylcholine receptor subunits influence the efficacy and potency of nicotine.

The first three transmembrane segments (M1-M3) of human nicotinic acetylcholine receptors (nAChRs) have been implicated in determining the efficacy of nicotine by studies of alpha3/alpha4 subunit chimeras. Nicotine has full efficacy on the alpha4beta2 nAChR and partial efficacy on the alpha3beta2 nAChR. Now, we have exchanged individually three amino acids between the alpha4 and the alpha3 subunits at positions 226(M1), 258(M2), and 262(M2). Also, similar exchanges were made in the beta2 and beta4 subunits at positions 224(M1), 226(M1), and 254(M2) (using alpha subunit numbering). Expression of these mutated nAChRs in Xenopus laevis oocytes showed that the mutated M1 amino acids were important in influencing the potency of ACh and nicotine. It is hypothesized that these M1 amino acids affect the stability between the resting and activated states of the nAChR. M2 amino acids altered the efficacy of nicotine, usually without altering its potency. When the residue located at position 258 in the M2 region of the alpha subunit was valine (as in the alpha3 subunit), the resulting nAChR exhibited partial efficacy for nicotine that was voltage-dependent. Therefore, we believe that these M2 amino acids contribute to the formation of a binding site for nicotine in the alpha3beta2 nAChR channel, which results in a low-affinity channel block, causing the lower efficacy of nicotine on this nAChR.

Spontaneous dissecting aneurysms of anterior and middle cerebral artery associated with brain infarction: A case report and review of the literature

BACKGROUND Intracranial dissecting aneurysms have been reported with increasing frequency and are recognized as a common cause of stroke. In some reviews and case reports, attempts have been made to compare the outcomes of surgical and medical treatments. However, the appropriate management of dissecting aneurysms in the anterior circulation remains controversial, especially in patients who also manifest cerebral infarction. CASE DESCRIPTION A 45-year-old male was diagnosed as having a dissecting aneurysm of the right middle cerebral artery (MCA) with cerebral infarction. In the course of conservative treatment, he developed a new cerebral infarction in the territory of the right anterior cerebral artery (ACA). Repeat cerebral angiograms revealed an increase in the aneurysmal dilatation of the right M2 and the appearance of a segmental dilatation of the right A2. He continued to be treated conservatively and his course was satisfactory. On subsequent angiograms, we observed resolution of the right A2 dissection and no further progression of the dilatation of the right M2. CONCLUSION This is the first reported case of simultaneous idiopathic dissecting aneurysms of different major arterial branches in the anterior circulation. Our review of the literature disclosed 36 and 23 cases, respectively, of dissecting aneurysms of the ACA and MCA. Many previously reported patients with these dissecting aneurysms involving subarachnoid hemorrhage (SAH) underwent surgery, which resulted in better outcome. More than half of the patients with ACA and MCA dissecting aneurysms had cerebral infarction. All ACA dissecting aneurysms involving ischemia occurred in the A2 region. The outcomes of both surgical and conservative management were equally satisfactory. On the other hand, in patients with MCA dissecting aneurysms, the area of ischemia frequently involved the M1 region; in these patients, conservative treatment resulted in poor outcomes. Therefore, revascularization distal to the compromised artery should be considered in patients with MCA-dissecting aneurysms who have ischemia. Careful interpretation of serial angiograms and/or magnetic resonance (MR) images is necessary because of the possibility of disease progression. If the aneurysmal size increases or there is progression of ischemic symptoms in the course of conservative treatment, surgery must be urgently evaluated.

Channel blockers acting at N-methyl-D-aspartate receptors: differential effects of mutations in the vestibule and ion channel pore.

A large number of structurally diverse compounds act as open-channel blockers of NMDA receptors. They may share discrete or overlapping binding sites within the channel. In this study, the effects of mutations in and around the membrane-spanning and pore-forming regions of NMDA receptor subunits were studied with three blockers, MK-801, memantine, and TB-3-4, using recombinant NMDA receptors expressed in Xenopus laevis oocytes. Mutations at the critical asparagine residues in the M2 loop of NR1 and NR2B and at a tryptophan residue in M2 of NR2B reduced block by MK-801, memantine, and TB-3-4. Mutations at residues in the pre-M1, M1, M3, post-M3, and post-M4 regions had differential effects on the three blockers. Many mutations in these regions reduced block by MK-801 and TB-3-4 but had no effect on block by memantine. The differential effects on block by memantine and MK-801 are unlikely to be caused by differences in the size of these blockers. Benzyl rings in MK-801 and TB-3-4 may make hydrophobic interactions with aromatic and hydrophobic amino acid residues in the pore. Some mutations in the pre-M1 and M3 regions generated constitutively open channels, characterized by large holding currents. The effects of the various mutants are discussed in the context of models based on the known structure of the pore of the KcsA potassium channel and on previous studies dealing with solvent accessible residues in NMDA receptor subunits as determined by modification after cysteine mutagenesis.

Motor cortex and the distributed anatomy of finger movements.

Voluntary movements are thought to be controlled via a well-ordered, spatially discrete, somatotopic map in the primary motor cortex (M1). We examined this hypothesis in monkeys trained to perform visually-cued, individuated flexion and extension movements of each digit and of the wrist. Single neurone recordings in M1 during such finger movements revealed two unexpected features. First, single M1 neurones often discharge during instructed movements of multiple digits. Second, neurones active during any particular instructed movement are distributed widely throughout the same M1 territory as neurones active during any other movement. Reversible, partial inactivation of the M1 hand representation produced by injection of 5-10 microg muscimol at one site impaired the monkeys' ability to perform finger movements, but no relationship was evident between the particular finger movements that were affected and the mediolateral location of the injection site along the central sulcus. Thus each finger movement is represented by activity distributed widely in the M1 upper extremity representation. If not controlled from spatially segregated M1 regions, movements of different fingers might be controlled by groups of spatially scattered but physiologically similar neurones. Cluster analysis of M1 neurones demonstrated a large group that discharged during most finger movements, and a small group that paused during most movements. Distinct functional groups of M1 neurones that might control particular finger movements were identified inconsistently. We therefore hypothesize that M1 neurones are a very diverse network controlling finger movements.

Stereoselective synthesis, in vitro, and initial in vivo evaluation of 1-methylpiperidin-4-yl α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenylacetate (IPIP): a novel radioiodinated molecular probe with high affinity for the muscarinic receptor

1-Methylpiperidin-4-yl α-hydroxy-α-(1-iodo-1-propen-3-yl)-α-phenylacetate (IPIP, Fig. 1) was investigated as a potential radioiodinated molecular probe targeted to the muscarinic receptor complex. The IPIP stereoisomers were synthesized via a chiral intermediate in >95% enantiomeric excess. The R-isomers demonstrated a M1 to M2 subtype selectivity of approximately 3 to 1 and the S-isomers demonstrated non-subtype selective binding in vitro. IPIP was radiolabeled with iodide-125 with an average radiochemical yield of 74.4% (±14.8, n = 5), specific activities >800 mCi/μmol, and radiochemical purities >97%. In vivo the Z-isomers demonstrated high uniform cerebral uptake suggesting non-subtype selective binding. In contrast, E-R-IPIP, after allowing a low uptake in M2 rich areas to clear, demonstrated a retention of activity in M1 and M4 rich cerebral regions. In addition, the cerebral uptake of E-R-IPIP and Z-S-IPIP were inhibited by 70–90% via pretreatment with R-QNB, an established muscarinic antagonist. An ex vivo metabolism study demonstrated Z-S-IPIP was stable at the receptor site with an absence of radiolabeled metabolites.

Genetic dissection of the Streptococcus pyogenes M1 protein: regions involved in fibronectin binding and intracellular invasion

Entry of serotype M1 Streptococcus pyogenes into host cells depends on binding of the host glycoprotein fibronectin (Fn) by the bacterial M1 protein. The present study was undertaken to localize the Fn binding region in M1 and assess other potential functions of M1. A set of recombinant M1 protein fragments were assayed for their capacities to bind Fn and inhibit ingestion of streptococci by epithelial cells. M1 protein, M6 protein and internally-deleted derivatives of M1 were expressed on the surface of Lactococcus lactis. Lactococci that expressed M1 or M6 protein bound Fn and were efficiently taken up by epithelial cells. Deletion of both the N-terminal A and B repeats regions of M1 abrogated Fn binding and intracellular invasion. Deletion of either the A domain (M1ΔA) or B repeats (M1ΔB) significantly reduced, but did not completely eliminate, Fn binding indicating that M1 protein may possess two independent Fn binding sites. Fn binding by the M1ΔA or M1ΔB proteins was insufficient for efficient invasion, however, suggesting that M protein binding alters the structure of Fn that, in turn, affects the interaction between Fn and epithelial cells. Although expression of M1, M6 or M1ΔB proteins led to aggregation of lactococcal cells, aggregation did not significantly contribute to invasion efficiency.

Structural modifications in the membrane-bound regions of the gastric H+/K+-ATPase upon ligand binding.

Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.

Requirement of Multiple Protein Domains and Residues for GatingKATP Channels by Intracellular pH

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.

Modifications in the CD36 binding domain of the Plasmodium falciparum variant antigen are responsible for the inability of chondroitin sulfate A adherent parasites to bind CD36

AbstractAdhesion of mature Plasmodium falciparum parasitized erythrocytes to microvascular endothelial cells or to placenta contributes directly to the virulence and severe pathology of P falciparum malaria. Whereas CD36 is the major endothelial receptor for microvasculature sequestration, infected erythrocytes adhering in the placenta bind chondroitin sulfate A (CSA) but not CD36. Binding to both receptors is mediated by different members of the large and diverse protein family P falciparum erythrocyte membrane protein-1 (PfEMP-1) and involves different regions of the molecule. The PfEMP-1–binding domain for CD36 resides in the cysteine-rich interdomain region 1 (CIDR-1). To explore why CSA-binding parasites do not bind CD36, CIDR-1 domains from CD36- or CSA-binding parasites were expressed in mammalian cells and tested for adhesion. Although CIDR-1 domains from CD36-adherent strains strongly bound CD36, those from CSA-adherent parasites did not. The CIDR-1 domain has also been reported to bind CSA. However, none of the CIDR-1 domains tested bound CSA. Chimeric proteins between CIDR-1 domains that bind or do not bind CD36 and mutagenesis experiments revealed that modifications in the minimal CD36-binding region (M2 region) are responsible for the inability of CSA-selected parasites to bind CD36. One of these modifications, mapped to a 3–amino acid substitution in the M2 region, ablated binding in one variant and largely reduced binding of another. These findings provide a molecular explanation for the inability of placental sequestered parasites to bind CD36 and provide additional insight into critical residues for the CIDR-1/CD36 interaction.

Embolization of calcific thrombi after tissue plasminogen activator treatment

Background and purpose: Embolic stroke has been reported after thrombolysis in cardiac patients but has not yet been documented after thrombolytic therapy for acute ischemic stroke. Description of cases: Patient 1 had a calcific embolus in the right M1 region on head computed tomography (CT) scan when treated with tissue plasminogen activator (tPA). Repeat imaging within hours showed distal migration of calcific fragments into the M2 region. Patient 2 had a calcific embolus in the right M1 region, as well as distal calcific emboli in multiple vascular distributions on initial head CT scan. She was treated with intravenous tPA but became unresponsive within 2 hours. Repeat imaging showed new calcium-density signal in the basilar artery. Conclusions: We present 2 cases of radiographically evident, calcific embolization after tPA therapy for acute ischemic stroke. Emboli with a calcific component may lyse with tPA, but such patients should be carefully monitored for distal or recurrent embolization. Copyright © 2001 by National Stroke Association

Pregnenolone sulfate block of GABA(A) receptors: mechanism and involvement of a residue in the M2 region of the alpha subunit.

Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the [gamma]-aminobutyric acid A (GABA(A)) receptor. We analysed the actions of PS on single-channel currents from recombinant GABA(A) receptors formed from [alpha]1, [beta]2 and [gamma]2L subunits. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 microM) and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel activity at either concentration of GABA. PS had no discernable effect on rapid processes: no effects were apparent on channel opening and closing, nor on GABA affinity, and a rapidly recovering desensitised state was not affected. Instead, PS produced a slowly developing block which occurred at a similar rate for receptors with open or closed channels and with one or two bound GABA molecules. The rate of block was independent of membrane potential, implying that the charged sulfate moiety does not move through the membrane field. Change in a specific residue near the intracellular end of the channel lining portion of the [alpha]1 subunit had a major effect on the rate of block. Mutation of the residue [alpha]1 V256S reduced the rate of block by 30-fold. A mutation at the homologous position of the [beta]2 subunit ([beta]2 A252S) had no effect, nor did a complementary mutation in the [gamma]2L subunit ([gamma]2L S266A). It seems likely that this residue is involved in a conformational change underlying block by PS, instead of forming part of the binding site for PS.

A Single Amino-Acid in the TM1 Domain Is an Important Determinant of the Desensitization Kinetics of Recombinant Human and Guinea Pig α-Homomeric 5-Hydroxytryptamine Type 3 Receptors

Desensitization of ligand-gated ion channels shapes synaptic responses and provides critical neuroprotection at central synapses, yet the molecular mechanisms underlying the desensitization process are poorly understood. Using the whole-cell voltage-clamp technique, we investigated desensitization kinetics of recombinant human and guinea pig alpha-homomeric 5-hydroxytryptamine type 3 (5-HT(3A)) receptors heterologously expressed in human embryonic kidney 293 cells. Human 5-HT(3A) receptors desensitize 3.5 times faster than does the homologous receptor from guinea pigs. By constructing various chimeras and through site-directed mutagenesis, we have identified a single serine in the M1 region of the human 5-HT(3A) receptor sequence (S248) that, when substituted with threonine found in the equivalent guinea pig sequence (T254), conferred guinea pig-like kinetics on the time course of desensitization of the human receptor. Correspondingly, the reverse mutation (guinea pig T254S) resulted in a fast, human-like time constant of desensitization. Thus, the primary structure of the M1 region is an important determinant of desensitization kinetics of recombinant 5-HT(3A) receptors.

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance.

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen-binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.