Domains determining ligand specificity for Ca2+ receptors.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the level of extracellular Ca2+. The Ca2+ receptor is a member of a family of G protein-coupled receptors that includes metabotropic glutamate receptors (mGluRs), gamma-aminobutyric acidB receptors, and putative pheromone receptors. As a family, these receptors are characterized by limited sequence homology and an unusually large putative extracellular domain (ECD). The ECD of the mGluRs is believed to determine agonist selectivity, but the functions of the structural domains of the Ca2+ receptor are not known. To identify structural determinants for cation recognition and activation of the Ca2+ receptor (and to further study the mGluRs), two chimeric receptors were constructed in which the large ECD of the Ca2+ receptor and the mGluR1 were interchanged. When expressed in Xenopus laevis oocytes, one of these chimeras, named CaR/mGluR1 [ECD of the Ca2+ receptor and transmembrane domain (TMD) of the mGluR1], responded to cation agonists (Gd3+, Ca2+, neomycin) of the Ca2+ receptor at concentrations similar to those necessary for activation of the native Ca2+ receptor. A reciprocal construct, named mGluR1/CaR (ECD of the mGluR1 and TMD of the Ca2+ receptor), was responsive to mGluR agonists but was much less sensitive to two of three cation agonists known to activate the Ca2+ receptor. A deletion construct of the Ca2+ receptor (DeltantCaR), which lacked virtually the entire ECD, was only activated by one of three agonists tested. These results suggest that the primary determinants for agonist activation of both the Ca2+ receptor and the mGluRs are found in the large ECD and that the Ca2+ receptor is possibly distinguished from the mGluRs in that it may contain sites in the TMD that permit activation by certain cation agonists.

Allosteric activation of the Ca2+ receptor expressed in Xenopus laevis oocytes by NPS 467 or NPS 568.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+ concentration was raised above 1.5 mM, whereas Ca2+ concentrations > 3 mM were generally necessary to elicit responses in oocytes expressing the human Ca2+ receptor. NPS 467 and NPS 568 potentiated the activation of ICl by extracellular Ca2+ in oocytes expressing either Ca2+ receptor homolog, and this resulted in a leftward shift of the Ca2+ concentration-response curve. Neither compound was active in the absence of extracellular Ca2+. Certain inorganic and organic cations known to activate the Ca2+ receptor were substituted for elevated levels of extracellular Ca2+ to increase ICl and the effects of these agonists were also potentiated by NPS 568 or NPS 467. The effects of NPS 568 were stereoselective and the R-enantiomer was about 10-fold more potent than the corresponding S-enantiomer. Neither NPS 467 nor 568 affected ICl in water-injected oocytes or in oocytes expressing the substance K receptor or the metabotropic glutamate receptor 1a. These results provide compelling evidence that NPS 467 and NPS 568 act directly upon the parathyroid Ca2+ receptor to increase its sensitivity to activation by extracellular Ca2+. This activity suggests that these compounds are positive allosteric modulators of the Ca2+ receptor. As such, these compounds define a new class of pharmacological agents with potent and selective actions on the Ca2+ receptor.

Colicin E1 binding to membranes: time-resolved studies of spin-labeled mutants.

To investigate the mechanism of interaction of the toxin colicin E1 with membranes, three cysteine substitution mutants and the wild type of the channel-forming fragment were spin labeled at the unique thiol. Time-resolved interaction of these labeled proteins with phospholipid vesicles was investigated with stopped-flow electron paramagnetic resonance spectroscopy. The fragment interacts with neutral bilayers at low pH, indicating that the interaction is hydrophobic rather than electrostatic. The interaction occurs in at least two distinct steps: (i) rapid adsorption to the surface; and (ii) slow, rate-limiting insertion of the hydrophobic central helices into the membrane interior.

FASTRUN: a special purpose, hardwired computer for molecular simulation.

We describe the design, construction, and performance of a special purpose, hardwired accelerator for molecular mechanical calculations called FASTRUN. The processor was designed at Columbia University in 1984, constructed in the Instrumentation Division of Brookhaven National Laboratory, and delivered to Columbia in final form in 1989. It was rendered functional for molecular mechanics in early 1990. Together with its host Star array processor, FASTRUN has a measured performance for molecular dynamics simulations which compares favorably with present day supercomputers. The hardware replication cost of FASTRUN is on the order of $100,000.00.

A single tryptic fragment of colicin E1 can form an ion channel: stoichiometry confirms kinetics.

The molecularity of the ion channel formed by peptide fragments of colicin has taken on particular significance since the length of the active peptide has been shown to be less than 90 amino acids and the lumen size at least 8 A. Cell survival experiments show that killing by colicin obeys single-hit statistics, and ion leakage rates from phospholipid vesicles are first order in colicin concentration. However, interpretation in molecular terms is generally complicated by the requirement of large numbers of colicin molecules per cell or vesicle. We have measured the discharge of potential across membranes of small phospholipid vesicles by following the changes in binding of potential sensitive spin labeled phosphonium ions as a function of the number of colicin fragments added. Because of the sensitivity of the method, it was possible to reliably investigate the effect of colicin in a range where there was no more than 0.2 colicins per vesicle. The quantitative results of these experiments yield a direct molecular stoichiometry and demonstrate that one C-terminal fragment of the colicin molecule per one vesicle is sufficient to induce a rapid ion flux in these vesicles. In addition, the experiments confirm earlier findings that the colicin fragments do not migrate from one vesicle to another at pH 4.5. Similar results are obtained with large unilamellar vesicles.

CARTOS II semi-automated nerve tracing: three-dimensional reconstruction from serial section micrographs.

The paper describes the history of tracing activities in the Levinthal lab from 1965 to the present. It also focuses on the evolution of electron micrograph (EM) tracing from early hand tracing reconstruction to the current, nearly fully automated CARTOS II tracing system. The present hardware systems and tracing algorithms are described in detail. Also presented are the partial tracing results of one EM data set in two and three dimensions.

Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation.

Colicin E1 is an E. coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel. We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398-406) within the C-terminal domain of the water-soluble form of the protein was investigated and exhibited an electron paramagnetic resonance (EPR) spectral periodicity strongly suggesting an amphiphilic alpha-helix. After removal of the N-terminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.

Infancy and early childhood follow-up of neonates with periventricular or intraventricular hemorrhage or isolated ventricular dilation: a case controlled study.

Survivors of periventricular or intraventricular hemorrhage and isolated ventricular dilation showed a higher incidence of major developmental problems in the motor areas than matched control subjects in infancy. This effect is still seen, but less evident in early childhood. Problems were mainly related to grades III and IV periventricular or intraventricular hemorrhage and isolated ventricular dilation. The outcome of newborns with grades I and II hemorrhage was benign.

A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene.

To search for genes involved in determining the morphology of individual neuronal types, a cDNA library was constructed from postnatal day 13 mouse cerebellum. From this library, 2 clones, L7 and L19, were isolated by a differential hybridization procedure and shown by in situ hybridization to be Purkinje cell-specific within the cerebellum. Both RNAs appear between postnatal days 4 and 8 and continue into adulthood, coinciding with terminal differentiation of the Purkinje cells. L7 seems to be expressed exclusively in the cerebellum, whereas L19 is expressed throughout the brain. Consistent with the RNA localization, L7 protein is found only in the cerebellum and is confined to the Purkinje cells. The L7 amino acid sequence has been deduced from the cDNA sequence, and a pseudo-repeat within the L7 protein sequence is homologous to the amino acids sequence in the primary translation product of the gene for human sis/PDGF.

Predicting antibody hypervariable loop conformations. II: Minimization and molecular dynamics studies of MCPC603 from many randomly generated loop conformations.

We describe a method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions [CDRs] in the crystallographically determined structure of MCPC603. The method is based on the generation of a large number of randomly generated conformations for the backbone of the loop being studied, followed by either minimization or molecular dynamics followed by minimization starting from these random structures. The details of the algorithm for the generation of the loops are presented in the first paper in this series (Shenkin et al. [submitted]). The results of minimization and molecular dynamics applied to these loops is presented here. For the two shortest CDRs studied (H1 and L2, which are five and seven amino acids long), minimizations and dynamics simulations which ignore interactions of the loop amino acids beyond the carbon beta replicate the conformation of the crystal structure closely. This suggests that these loops fold independently of sequence variation. For the third CDR (L3, which is nine amino acids), those portions of the CDR near its base which are hydrogen bonded to framework are well replicated by our procedures, but the top of the loop shows significant conformational variability. This variability persists when side chain interactions for the MCPC603 sequence are included. For a fourth CDR (H3, which is 11 amino acids long), new low-energy backbone conformations are found; however, only those which are close to the crystal are compatible with the sequence when side chain interactions are taken into account. Results from minimization and dynamics on single CDRs with all other CDRs removed are presented. These allow us to explore the extent to which individual CDR conformations are determined by interactions with framework only.

A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1.

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain.

C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20 kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments. Apparently trypsin removes segments of protein N-terminal to the channel-forming region, since gating behavior of the shortest fragment is little affected by the enzyme. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. Trypsin modifies only closed channels from the cis side (the side to which protein has been added) and only open channels from the trans side. These results suggest that regions outside the channel-forming domain affect ion selectivity and gating, and they also provide evidence that large protein segments outside the channel-forming domain are translocated across the membrane with channel gating.

A graph theory model of the glomerular capillary network and its development.

Graph theory methods were used to analyze the topology of the renal glomerular capillary network using data both from a serial reconstruction of a rat glomerulus and from the literature. The graphs obtained were tested for planarity, and all but one were found to be nonplanar. This result indicated that the development of the glomerular capillary network must include a nonplanar growth process, and new growth models were proposed. In addition, the statistical properties of capillary branching patterns were analyzed, and a node degree distribution function estimate was obtained.