RESUMO
Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Engenharia de Proteínas/métodos , Receptor Muscarínico M2/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acetilcolina/farmacologia , Regulação Alostérica , Animais , Dopamina/farmacologia , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusão/metabolismo , XenopusRESUMO
Nanoscale electrical biosensors are promising tools for diagnostics and high-throughput screening systems. The electrical signal allows label-free assays with a high signal-to-noise ratio and fast real-time measurements. The challenge in developing such biosensors lies in functionally connecting a molecule detector to an electrical switch. Advances in this field have relied on synthetic ion-conducting pores and modified ion channels that are not yet suitable for biomolecule screening. Here we report the design and characterization of a novel bioelectric-sensing platform engineered by coupling an ion channel, which serves as the electrical probe, to G-protein-coupled receptors (GPCRs), a family of receptors that detect molecules outside the cell. These ion-channel-coupled receptors may potentially detect a wide range of ligands recognized by natural or altered GPCRs, which are known to be major pharmaceutical targets. This could form a unique platform for label-free drug screening.