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Biomedical subjects

D A Dougherty

Publications and source records attributed to D A Dougherty.

At least 37 records · Page 2Linked to original sources
PubMed1998

Flash decaging of tyrosine sidechains in an ion channel.

A nonsense codon suppression technique was employed to incorporate ortho-nitrobenzyl tyrosine, "caged tyrosine," in place of tyrosine at any of three positions (93, 127, or 198) in the alpha subunit of the muscle nicotinic ACh receptor (nAChR) expressed in Xenopus oocytes. The ortho-nitrobenzyl group was then removed by 1 ms flashes at 300-350 nm to yield tyrosine itself while macroscopic currents were recorded during steady ACh exposure. Responses to multiple flashes showed (1) that each flash decages up to 17% of the tyrosines and (2) that two tyrosines must be decaged per receptor for a response. The conductance relaxations showed multiple kinetic components; rate constants (<0.1 s(-1) to 10(3) s(-1)) depended on pH and the site of incorporation, and relative amplitudes depended on the number of prior flashes. This method, which is potentially quite general, (1) provides a time-resolved assay for the behavior of a protein when a mutant sidechain is abruptly changed to the wild-type residue and (2) will also allow for selective decaging of sidechains that are candidates for covalent modification (such as phosphorylation) in specific proteins in intact cells.

Acetylcholine↗
PubMed1998

Stereochemical issues in studies of ion channel proteins.

Studies of ion channel proteins present many special challenges, including the potential for novel stereochemical phenomena. Here we describe several examples in which modern studies of ion channels have involved or made use of stereochemistry.

Animals↗
PubMed1997

Expression and circular dichroism studies of the extracellular domain of the alpha subunit of the nicotinic acetylcholine receptor.

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.

Amino Acid Sequence↗
PubMed1997

Site-specific, photochemical proteolysis applied to ion channels in vivo.

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.

Animals↗
PubMed1997

Site-specific incorporation of biotinylated amino acids to identify surface-exposed residues in integral membrane proteins.

BACKGROUND: A key structural issue for all integral membrane proteins is the exposure of individual residues to the intracellular or extracellular media. This issue involves the basic transmembrane topology as well as more subtle variations in surface accessibility. Direct methods to evaluate the degree of exposure for residues in functional proteins expressed in living cells would be highly valuable. We sought to develop a new experimental method to determine highly surface-exposed residues, and thus transmembrane topology of membrane proteins expressed in Xenopus oocytes. RESULTS: We have used the in vivo nonsense suppression technique to incorporate biotinylated unnatural amino acids into functional ion channels expressed in Xenopus oocytes. Binding of 125I-streptavidin to biotinylated receptors was used to determine the surface exposure of individual amino acids. In particular, we studied the main immunogenic region of the nicotinic acetylcholine receptor. The biotin-containing amino acid biocytin was efficiently incorporated into five sites in the main immunogenic region and extracellular streptavidin bound to one residue in particular, alpha 70. The position of alpha 70 as highly exposed on the receptor surface was thus established. CONCLUSIONS: The in vivo nonsense suppression technique has been extended to provide the first in a potential series of methods to identify exposed residues and to assess their relative exposure in functional proteins expressed in Xenopus oocytes.

Amino Acids↗
PubMed1996

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation.

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G beta gamma subunits, but instead shows constitutive activation, and (ii) is no longer a K(+)-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na(+)-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

Acetylcholine↗
PubMed1996

Subunit stoichiometry of a heteromultimeric G protein-coupled inward-rectifier K+ channel.

We investigated the stoichiometry of the heteromultimeric G protein-coupled inward-recitfier K+ channel (GIRK) formed from GIRK1 and GIRK4 subunits. Multimeric GIRK constructs with several concatenated channel subunits were expressed in Xenopus oocytes. Coexpression of various trimeric constructs with different monomers clearly showed that the functional channel has stoichiometry (GIRK1)2(GIRK4)2. Efforts to establish a preferred arrangement of subunits around the channel pore suggest that more than one arrangement may be viable.

Animals↗
PubMed1996

Cation-pi interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide.

The cation-pi interaction is an important, general force for molecular recognition in biological receptors. Through the sidechains of aromatic amino acids, novel binding sites for cationic ligands such as acetylcholine can be constructed. We report here a number of calculations on prototypical cation-pi systems, emphasizing structures of relevance to biological receptors and prototypical heterocycles of the type often of importance in medicinal chemistry. Trends in the data can be rationalized using a relatively simple model that emphasizes the electrostatic component of the cation-pi interaction. In particular, plots of the electrostatic potential surfaces of the relevant aromatics provide useful guidelines for predicting cation-pi interactions in new systems.

Benzene↗
PubMed1996

An engineered Tetrahymena tRNAGln for in vivo incorporation of unnatural amino acids into proteins by nonsense suppression.

A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.

Acetylcholine↗
PubMed1996

Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp.

Cations bind to the pi face of an aromatic structure through a surprisingly strong, non-covalent force termed the cation-pi interaction. The magnitude and generality of the effect have been established by gas-phase measurements and by studies of model receptors in aqueous media. To first order, the interaction can be considered an electrostatic attraction between a positive charge and the quadrupole moment of the aromatic. A great deal of direct and circumstantial evidence indicates that cation-pi interactions are important in a variety of proteins that bind cationic ligands or substrates. In this context, the amino acids phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) can be viewed as polar, yet hydrophobic, residues.

Acetylcholine↗
PubMed1996

Determinants of nicotinic receptor gating in natural and unnatural side chain structures at the M2 9' position.

A nonsense suppression method was employed to incorporate a total of four natural and six unnatural residues at the 9' position of the M2 region in the beta, gamma, and delta subunits of muscle nicotinic receptors. In 33 pairwise comparisons of functional properties as influenced by structural features including side chain length, branching, and substitution of oxygen for methylene carbons, it is concluded that increased polarity in the side chains at the 9' position consistently increases the sensitivity to acetylcholine. In addition, the stereochemistry of the side chain can have marked influences on the EC50, primarily because of changes in the single-channel open time. For the case of isoleucine versus allo-isoleucine in the delta subunit, these changes are themselves modified by mutations at the 9' position in other subunits. The data suggest an especially strong interaction between the beta and delta subunits in the pore region, leading in turn to a suggested arrangement of subunits within the pentamer.

Animals↗
PubMed1996

Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: tests with novel side chains and with several agonists.

Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, alpha 93, alpha 190, and alpha 198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of alpha Tyr93, whereas a variety of substituents are functional at the analogous position of alpha Tyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

Amino Acids↗
PubMed1993

A mechanism for ion selectivity in potassium channels: computational studies of cation-pi interactions.

A combination of computational methods has been used to evaluate the interaction between the pi face of a benzene molecule and the monovalent cations of lithium, sodium, potassium, and rubidium. In the gas phase, the ions are strongly bound, and the affinity for benzene follows the expected electrostatic trend (lithium, largest; rubidium, smallest). However, in an aqueous environment, a reordering occurs such that the potassium ion is preferred over all the other ions for 2:1 benzene:ion complexes. The selectivity sequence parallels that seen in voltage-gated potassium channels. Given that several conserved aromatic residues are present in the pore region of such channels, these results suggest that the cation-pi interaction may be responsible for the ion selectivity in potassium channels.

Amino Acid Sequence↗
PubMed1993

Effect of U88999E on experimental cerebral vasospasm in rabbits.

U88999E, 7-[4,(4, 4'-difluorobenzhydryl)piperazino-1-methyl]-4- isopropyl-2-methoxy-2,4,6-cycloheptatrien-1-one hydrochloride, is a recently developed tropolone derivative that inhibits lipid peroxidation and also acts as a calcium antagonist. The effects of U88999E on basilar artery tone were examined in two model systems: 1) an in vitro preparation of arterial rings that measures isometric tension, and 2) an in vivo model of cerebral vasospasm measuring arterial diameter. U88999E elicited dose-dependent relaxation of preconstricted arterial rings maintained in vitro. Ring preparations were preconstricted using elevated potassium (40 mmol/L), uridine triphosphate (10(-3) mol/L), or endothelin-1 (10(-8) mol/L); U88999E reversed these constrictions across a concentration range of 10(-8) to 10(-5) mol/L. The potency of U88999E for relaxing preconstricted vessels was slightly less than that observed for flunarizine or diltiazem. A dose-dependent, relaxing effect of U88999E on potassium-induced contractions was observed in the presence of calcium concentrations ranging from 0.03 to 20 mmol/L. Vasospasm of basilar arteries after subarachnoid hemorrhage was inhibited in a dose-dependent and significant manner by intravenous injections of U88999E. Animals receiving intraperitoneal injections of U88999E also exhibited a tendency for reduced vasospasm; however, this effect did not achieve statistical significance. These findings suggest that U88999E may be useful in the prevention of cerebral vasospasm after subarachnoid hemorrhage.

Animals↗