-
[show abstract]
[hide abstract]
ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory
neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects
in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally
related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders,
and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the
ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the
residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor
binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB)
of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed
by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site
has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π
interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.
Journal of Biological Chemistry 04/2011; 286(16):14618-14627. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.
Journal of Biological Chemistry 02/2011; 286(16):14618-27. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nicotine addiction begins with high-affinity binding of nicotine to acetylcholine (ACh) receptors in the brain. The end result is over 4,000,000 smoking-related deaths annually worldwide and the largest source of preventable mortality in developed countries. Stress reduction, pleasure, improved cognition and other central nervous system effects are strongly associated with smoking. However, if nicotine activated ACh receptors found in muscle as potently as it does brain ACh receptors, smoking would cause intolerable and perhaps fatal muscle contractions. Despite extensive pharmacological, functional and structural studies of ACh receptors, the basis for the differential action of nicotine on brain compared with muscle ACh receptors has not been determined. Here we show that at the alpha4beta2 brain receptors thought to underlie nicotine addiction, the high affinity for nicotine is the result of a strong cation-pi interaction to a specific aromatic amino acid of the receptor, TrpB. In contrast, the low affinity for nicotine at the muscle-type ACh receptor is largely due to the fact that this key interaction is absent, even though the immediate binding site residues, including the key amino acid TrpB, are identical in the brain and muscle receptors. At the same time a hydrogen bond from nicotine to the backbone carbonyl of TrpB is enhanced in the neuronal receptor relative to the muscle type. A point mutation near TrpB that differentiates alpha4beta2 and muscle-type receptors seems to influence the shape of the binding site, allowing nicotine to interact more strongly with TrpB in the neuronal receptor. ACh receptors are established therapeutic targets for Alzheimer's disease, schizophrenia, Parkinson's disease, smoking cessation, pain, attention-deficit hyperactivity disorder, epilepsy, autism and depression. Along with solving a chemical mystery in nicotine addiction, our results provide guidance for efforts to develop drugs that target specific types of nicotinic receptors.
Nature 04/2009; 458(7237):534-7. · 36.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Biological Chemistry 01/2006; 280(50):41655-66. · 4.77 Impact Factor
-
Xinan Xiu
[show abstract]
[hide abstract]
ABSTRACT: NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Nicotinic acetylcholine receptors (nAChR) are an important family of ligand gated ion channels found throughout the CNS and the PNS. They have been indicated in a series of physiological functions and pathological states. nAChRs have received extensive study in the past as a prototype of the Cys loop LGIC member. Growing interest in developing subtype specific agents targeting nAChRs to treat neurological diseases require more detailed structural and functional information in the numerous members of the nAChR family. We performed structure-function studies on the chemical scale of several of the most important members of this family using a powerful combination of conventional mutagenesis and unnatural amino acid incorporations. Chapter 2 describes our research in studying the channel gating mechanism of the prototypic nAChR, the muscle type [...]. We studied thoroughly the gating interface of the receptor and 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. Chapter 3 reports our studies in the ligand binding mechanism of the most prevalent neuronal type [...] and α7 nAChR. We identified a cation-π interaction and a hydrogen bond employed by nicotine with the [...] receptor. These two key interactions are absent or significantly diminished in both the muscle type receptors and in the α7 form of neuronal receptor. In Chapter 4 we studied the ligand binding mechanism of a relatively newly characterized neuronal receptor [...]. From these studies, we found that in the Cys loop superfamily, homology in amino acid sequences and structures do not translate into a shared functional mechanism. In fact, different sets of chemical interactions are adopted between ligands and the receptor, and between amino acids within the ion channel proteins, both in ligand binding and channel gating. Ion channels are membrane bound multi-subunit macromolecules. We are able to carry out such exhaustive detailed structure-function studies by means of the fast developing methodology of unnatural amino acid incorporation by nonsense suppression. This thesis also describes our effort to improve the efficiency of nonsense suppression. In particular, we designed multiple 21nt small interfering RNA (siRNA) targeting release factor 1 (eRF1) in both HEK cells and Xenopus oocytes, and monitored the nonsense suppression efficiency change in vivo and in vitro by RNA PCR, Western blotting, fluorescence, and electrophysiology (Chapter 5).
-
[show abstract]
[hide abstract]
ABSTRACT: Nicotine addiction begins with high-affinity binding of nicotine to
acetylcholine (ACh) receptors in the brain. The end result is over
4,000,000 smoking-related deaths annually worldwide and the
largest source of preventable mortality in developed countries.
Stress reduction, pleasure, improved cognition and other central
nervous system effects are strongly associated with smoking.
However, if nicotine activated ACh receptors found in muscle as
potently as it does brain ACh receptors, smoking would cause intolerable
and perhaps fatal muscle contractions. Despite extensive
pharmacological, functional and structural studies of ACh receptors,
the basis for the differential action of nicotine on brain compared
with muscle ACh receptors has not been determined. Here we
show that at the α4β2 brain receptors thought to underlie nicotine
addiction, the high affinity for nicotine is the result of a strong
cation–π interaction to a specific aromatic amino acid of the receptor,
TrpB. In contrast, the low affinity for nicotine at the muscle type ACh
receptor is largely due to the fact that this key interaction is
absent, even though the immediate binding site residues, including
the key amino acid TrpB, are identical in the brain and muscle
receptors. At the same time a hydrogen bond from nicotine to the
backbone carbonyl of TrpB is enhanced in the neuronal receptor
relative to the muscle type. A point mutation near TrpB that differentiates
α4β2 and muscle-type receptors seems to influence the
shape of the binding site, allowing nicotine to interact more strongly
with TrpB in the neuronal receptor. ACh receptors are established
therapeutic targets for Alzheimer’s disease, schizophrenia,
Parkinson’s disease, smoking cessation, pain, attention-deficit
hyperactivity disorder, epilepsy, autism and depression. Along with
solving a chemical mystery in nicotine addiction, our results provide
guidance for efforts to develop drugs that target specific types of
nicotinic receptors.
