Biomedical subjects

Dennis A Dougherty

Publications and source records attributed to Dennis A Dougherty.

At least 19 recordsLinked to original sources

PubMed2007

Chemical-scale studies on the role of a conserved aspartate in preorganizing the agonist binding site of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site but does not directly contact the ligand plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently, the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function) or if a compensating backbone mutation is introduced to relieve unfavorable electrostatics.

Amino Acid Sequence↗

PubMed2006

Reconstitution of ion channels in agarose-supported silicon orifices.

A silicon wafer with eight individually addressable microfabricated orifices was used for ion channel reconstitution and single-channel recording. A spin-on fluoropolymer created an insulating, hydrophobic interface that was more stable than silane. Total capacitance of the membranes was <10 pF, making it easy to evaluate bilayer formation by capacitance change. Orifices of 50-250 microm diameter were tested for ease and stability of bilayer formation; only those >100 microm resulted in ion channel function. Bilayers were formed over an agarose supporting layer by application of lipid in decane with a paintbrush; a second layer of agarose could then be added to stabilize the structure and prevent evaporation. Microfluidic wells were constructed on glass plates for ease of assembly and visualization of fluid flow, as well as high-resolution microscopy for studies using fluorescent lipids and channels. The microfluidics consisted of reversibly bonded silicone rubber (PDMS), so that the entire device could be washed and reused. Total electrical noise in the device was low enough to permit single-channel resolution. Successful channel insertions were observed with a self-assembling ionophore (alamethicin) as well as a complex, vesicle-associated mammalian channel (human glycine receptor, GlyR). A "hands-free" approach to bilayer formation was also tested, where lipid in solvent was applied to the wafer by spin-coating, dried, and then "sandwiched" between layers of agarose above and below the nitride. Electrical properties consistent with bilayers were observed and alamethicin recordings were obtained, however this method is not compatible with the fusion of vesicles containing mammalian channels.

Biomimetics↗

PubMed2006

In vivo incorporation of multiple unnatural amino acids through nonsense and frameshift suppression.

Site-specific incorporation of unnatural amino acids (UAAs) into proteins is a valuable tool for studying structure-function relationships, incorporating biophysical probes, and elucidating protein-protein interactions. In higher eukaryotic cells, the methodology is currently limited to incorporation of a single UAA in response to a stop codon, which is known as nonsense suppression. Frameshift suppression is a unique methodology for incorporating UAAs in response to quadruplet codons, but currently, it is mostly limited to in vitro protein translation systems. Here, we evaluate the viability of frameshift suppression in Xenopus oocytes. We demonstrate UAA incorporation by using yeast phenylalanine frameshift suppressor (YFFS) tRNAs that recognize two different quadruplet codons (CGGG and GGGU) in vivo. Suppression efficiency of the YFFS tRNAs increases nonlinearly with the amount of injected tRNA, suggesting a significant competition with endogenous, triplet-recognizing tRNA. Both frameshift suppressor tRNAs are less efficient than the amber suppressor tRNA THG73 (Tetrahymena thermophila G73), which has been used extensively for UAA incorporation in Xenopus oocytes. However, the two YFFS tRNAs are more "orthogonal" to the Xenopus system than THG73, and they offer a viable replacement when suppressing at promiscuous sites. To illustrate the potential of combining nonsense and frameshift suppression, we have site-specifically incorporated two and three UAAs simultaneously into a neuroreceptor expressed in vivo.

Amino Acids↗

PubMed2006

Probing the Mg2+ blockade site of an N-methyl-D-aspartate (NMDA) receptor with unnatural amino acid mutagenesis.

The N -methyl-D-aspartate (NMDA) receptor plays a central role in learning and memory in the mammalian CNS. At normal neuronal resting membrane potentials, the pore of this glutamate-gated ion channel is blocked by a Mg(2+) ion. Previous work suggests that the Mg(2+) binding site is quite novel, involving several asparagine residues and a cation-pi interaction between Mg(2+) and a conserved tryptophan in the pore. Using unnatural amino acid mutagenesis, we show that no such cation-pi interaction exists. The implicated tryptophan instead appears to play a structural role that can only be fulfilled by a rigid, flat, hydrophobic residue. This is the first demonstration of unnatural amino acid incorporation in the NMDA receptor, and it opens the way for future investigations of this pivotal neuroreceptor.

Amino Acid Substitution↗

PubMed2006

Voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance.

A detailed picture of water and ion properties in small pores is important for understanding the behavior of biological ion channels. Several recent modeling studies have shown that small, hydrophobic pores exclude water and ions even if they are physically large enough to accommodate them, a mechanism called hydrophobic gating. This mechanism has been implicated in the gating of several channels, including the mechanosensitive channel of small conductance (MscS). Although the pore in the crystal structure of MscS is wide and was initially hypothesized to be open, it is lined by hydrophobic residues and may represent a nonconducting state. Molecular dynamics simulations were performed on MscS to determine whether or not the structure can conduct ions. Unlike previous simulations of hydrophobic nanopores, electric fields were applied to this system to model the transmembrane potential, which proved to be important. Although simulations without a potential resulted in a dehydrated, occluded pore, the application of a potential increased the hydration of the pore and resulted in current flow through the channel. The calculated channel conductance was in good agreement with experiment. Therefore, it is likely that the MscS crystal structure is closer to a conducting than a nonconducting state.

Cell Membrane↗

PubMed2006

A cation-pi interaction between extracellular TEA and an aromatic residue in potassium channels.

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation-pi interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation-pi binding ability of an aromatic ring, progressively increased the inhibitory constant K(i) for the TEA block of Shaker. A larger increase in K(i) was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation-pi component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation-pi mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation-pi interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.

Amino Acid Sequence↗

PubMed2005

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel.

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.

Animals↗

PubMed2005

A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors.

In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on gamma-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.

Acetylcholine↗

PubMed2005

Caged phosphoproteins.

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.

Cyclic AMP-Dependent Protein Kinases↗

PubMed2005

Using physical chemistry to differentiate nicotinic from cholinergic agonists at the nicotinic acetylcholine receptor.

The binding of three distinct agonists-acetylcholine (ACh), nicotine, and epibatidine-to the nicotinic acetylcholine receptor has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp alpha149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond. A simple structural model that considers only possible interactions with Trp alpha149 suggests that a novel aromatic C-H...O=C hydrogen bond further augments the binding of epibatidine. These studies illustrate the subtleties and complexities of the interactions between drugs and membrane receptors and establish a paradigm for obtaining detailed structural information.

Acetylcholine↗

PubMed2005

A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor.

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.

Amino Acid Substitution↗

PubMed2004

Tyrosine residues that control binding and gating in the 5-hydroxytryptamine3 receptor revealed by unnatural amino acid mutagenesis.

The mechanism by which agonist binding triggers pore opening in ligand-gated ion channels is poorly understood. Here, we used unnatural amino acid mutagenesis to introduce subtle changes to the side chains of tyrosine residues (Tyr141, Tyr143, Tyr153, and Tyr234), which dominate the 5-HT3 receptor binding site. Heterologous expression in oocytes, combined with radioligand binding data and a model of 5-HT (serotonin) computationally docked into the binding site, has allowed us to determine which of these residues are responsible for binding and/or gating. We have shown that Tyr 143 forms a hydrogen bond that is essential for receptor gating but does not affect binding, whereas a hydrogen bond formed by Tyr153 is involved in both binding and gating of the receptor. The aromatic group of Tyr234 is essential for binding and gating, whereas its hydroxyl does not affect binding but plays a steric role in receptor gating. Tyr141 is not involved in agonist binding or receptor gating but is important for antagonist interactions. These data, combined with a new model of the nonliganded 5-HT3 receptor, lead to a mechanistic explanation of the interactions that initiate the conformational change leading to channel opening. Thus, we suggest that agonist entry into the binding pocket may displace Tyr143 and Tyr153 and results in their forming new hydrogen bonds. These bonds may form part of the network of bond rearrangements that trigger the conformational change leading to channel opening. Similar rearrangements may initiate gating in all Cys-loop receptors.

Amino Acids↗

PubMed2004

A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site.

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta 19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta F/F) approximately 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta F agreed well with those for epibatidine-induced currents, but were shifted approximately 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha gamma-binding site than to the alpha delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta F monitors an event linked to binding specifically at the alpha delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta F occurs within milliseconds, i.e., during activation. At low [ACh] (< or = 10 microM), a phase of Delta F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta F is complete before the slowest phase of recovery from desensitization (time constant approximately 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.

Acetylcholine↗

PubMed2004

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis.

Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed.

Electron Spin Resonance Spectroscopy↗

PubMed2004

Cys-loop receptors: new twists and turns.

New hypotheses and predictions have arisen from recent work revealing atomic-scale or near-atomic-scale structures of receptors in the 'Cys-loop' superfamily. How general is the cation-pi interaction between the natural ligand and a tryptophan residue in the aromatic box, and does this interaction extend to other ligands? What is the pathway from the binding site to gating, and what are the conformational changes during gating and desensitization? Is current flow through intracellular 'portals' in the wall of the channel a general feature? This article discusses these and related questions, emphasizing nicotinic ACh receptors and also discussing data from other members of this superfamily.

Animals↗

PubMed2003

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: electrophysiology-coordinated photochemistry and mass spectrometry.

We characterized the differential accessibility of the nicotinic acetylcholine receptor alpha1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or approximately 50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the beta8-beta9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128-142), the cytoplasmic loop (M3-M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane alpha-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3-M4 loop are near to the lipid bilayer.

Amino Acid Sequence↗

PubMed2003

Different binding orientations for the same agonist at homologous receptors: a lock and key or a simple wedge?

Using unnatural amino acid mutagenesis, the binding site for serotonin at the novel Caenorhabditis elegans receptor MOD-1 has been probed. As with the closely related serotonin receptor 5-HT3, MOD-1 makes use of a strong cation-pi interaction between the ammonium of serotonin and the indole side chain of a tryptophan. However, the specific Trp used by MOD-1 is different from that used for 5-HT3 (and the nAChR), aligning with a residue more than 40 amino acids distant in sequence space and on a different "loop" of the agonist binding site. This suggests a significant rearrangement of the ligand on binding these two closely related receptors. It is suggested that, unlike enzymes, receptors and other signaling molecules may need only to deliver an agonist to a general binding region, rather than establishing precise drug-receptor interactions.

Binding Sites↗

PubMed2003

Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL: implications for channel gating and evolutionary design.

Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function. Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified. Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function. The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer. The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels.

Amino Acid Sequence↗