No abstract

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.

Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8-β9 loop in the extracellular ligand-binding domain. The β8-β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8-β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.ligand-gated ion channel | X-ray crystallography | allosteric modulation | Cys-loop receptor | nicotinic acetylcholine receptor C hlorpromazine (CPZ) (Fig. 1), a phenothiazine-derived antipsychotic drug, was introduced in psychiatry in the early 1950s, revolutionizing the treatment of psychotic disorders (1, 2). The main mechanism of action of CPZ consists in the blockage of dopamine receptors (2-4), but the numerous side effects associated with this drug indicate that it interacts with other physiologically relevant targets. CPZ was indeed shown to interfere with several voltage-and ligand-gated channels: it inhibits neuronal voltage-gated K + channels (5-7), BK Ca channels (8), and the human α 1E subunit-mediated Ca 2+ channels (9); CPZ was also shown to inhibit GABAergic currents (10, 11), specifically through GABA A receptors (GABA A Rs) (12), and to inhibit serotonin type-3 receptors (5-HT 3 Rs) (13, 14) and nicotinic acetylcholine receptors (nAChRs) (15, 16), members of the Cys-loop receptor family.The Cys-loop receptor family is composed of membranespanning ligand-gated ion channels that are responsible for fast excitatory or inhibitory synaptic neurotransmission. They are composed of five identical or nonidentical subunits, each of them comprising an N-terminal extracellular domain, which contains the neurotransmitter binding site, four transmembrane helices, that when assembled allow ions to pass through the membrane, and an intracellular domain, responsible for channel conductance, receptor modulation, and trafficking (17, 18). Initial structural insight into the ...

Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a co-crystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing, selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine our previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. In order to validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogs of compound 5. The most promising compound 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral endpoints in the rodent studies.

Highlights d The LRRs of SDS22 interact with PP1 helices a5 and a6 d A basic surface patch of SDS22 binds phosphorylated BCLAF1 d Many PP1 holoenzymes contain SDS22 as a third subunit d SDS22 prevents the autodephosphorylation of PP1