RESUMO
Ca2+ antagonist drugs are widely used in therapy of cardiovascular disorders. Three chemical classes of drugs bind to three separate, but allosterically interacting, receptor sites on CaV1.2 channels, the most prominent voltage-gated Ca2+ (CaV) channel type in myocytes in cardiac and vascular smooth muscle. The 1,4-dihydropyridines are used primarily for treatment of hypertension and angina pectoris and are thought to act as allosteric modulators of voltage-dependent Ca2+ channel activation, whereas phenylalkylamines and benzothiazepines are used primarily for treatment of cardiac arrhythmias and are thought to physically block the pore. The structural basis for the different binding, action, and therapeutic uses of these drugs remains unknown. Here we present crystallographic and functional analyses of drug binding to the bacterial homotetrameric model CaV channel CaVAb, which is inhibited by dihydropyridines and phenylalkylamines with nanomolar affinity in a state-dependent manner. The binding site for amlodipine and other dihydropyridines is located on the external, lipid-facing surface of the pore module, positioned at the interface of two subunits. Dihydropyridine binding allosterically induces an asymmetric conformation of the selectivity filter, in which partially dehydrated Ca2+ interacts directly with one subunit and blocks the pore. In contrast, the phenylalkylamine Br-verapamil binds in the central cavity of the pore on the intracellular side of the selectivity filter, physically blocking the ion-conducting pathway. Structure-based mutations of key amino-acid residues confirm drug binding at both sites. Our results define the structural basis for binding of dihydropyridines and phenylalkylamines at their distinct receptor sites on CaV channels and offer key insights into their fundamental mechanisms of action and differential therapeutic uses in cardiovascular diseases.
Assuntos
Aminas/química , Aminas/farmacologia , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/química , Di-Hidropiridinas/química , Di-Hidropiridinas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Aminas/efeitos adversos , Anlodipino/química , Anlodipino/farmacologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Cálcio/química , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Linhagem Celular , Cristalografia por Raios X , Di-Hidropiridinas/efeitos adversos , Lipídeos/química , Modelos Moleculares , Mariposas , Mutação , Niacina/análogos & derivados , Niacina/química , Niacina/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Verapamil/química , Verapamil/farmacologiaRESUMO
Voltage-gated sodium channels initiate action potentials in nerve, muscle, and other electrically excitable cells. Voltage-gated calcium channels are activated by depolarization during action potentials, and calcium influx through them is the key second messenger of electrical signaling, initiating secretion, contraction, neurotransmission, gene transcription, and many other intracellular processes. Drugs that block sodium channels are used in local anesthesia and the treatment of epilepsy, bipolar disorder, chronic pain, and cardiac arrhythmia. Drugs that block calcium channels are used in the treatment of epilepsy, chronic pain, and cardiovascular disorders, including hypertension, angina pectoris, and cardiac arrhythmia. The principal pore-forming subunits of voltage-gated sodium and calcium channels are structurally related and likely to have evolved from ancestral voltage-gated sodium channels that are widely expressed in prokaryotes. Determination of the structure of a bacterial ancestor of voltage-gated sodium and calcium channels at high resolution now provides a three-dimensional view of the binding sites for drugs acting on sodium and calcium channels. In this minireview, we outline the different classes of sodium and calcium channel drugs, review studies that have identified amino acid residues that are required for their binding and therapeutic actions, and illustrate how the analogs of those key amino acid residues may form drug-binding sites in three-dimensional models derived from bacterial channels.
Assuntos
Canais de Cálcio/química , Mamíferos/metabolismo , Canais de Sódio/química , Potenciais de Ação , Animais , Sítios de Ligação , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/metabolismo , Humanos , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêutico , Canais de Sódio/metabolismo , Homologia Estrutural de ProteínaRESUMO
Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.
Assuntos
Química Click , Fosfatidilinositol 3-Quinases , Transporte Proteico , Receptores Proteína Tirosina Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Transdução de Sinais , Membrana Celular/metabolismo , Optogenética , Código Genético , Luz , Animais , Células HEK293RESUMO
Receptor tyrosine kinase signaling is characterized by complex webs of interconnected pathways that regulate diverse cellular functions. The complexity of signaling is a barrier to understanding the pathways that control any particular function. In this work, we use a novel combination of approaches and a new click chemistry probe to determine the role of one pathway in regulating cell surface expression of an ion channel and a receptor tyrosine kinase. We applied an optogenetic approach to uncouple activation of the PI3K pathway from other pathways downstream of RTK activation. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the PM in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.