Ca2+ antagonist drugs are widely used in therapy of cardiovascular disorders1,2. 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 muscle3–9. 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 pore1,2. 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.

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 threedimensional models derived from bacterial channels.

Once known best for its toxic effects, recent work has shown that hydrogen sulfide (H2S) has many roles as a cellular messenger. For example, both endogenously produced and exogenously supplied H2S protect against cellular damage and death associated with ischemia/reperfusion injury in mammals. The mechanistic relationship between beneficial and toxic effects of H2S is not understood. We have developed C. elegans as a model to understand the mechanistic basis of H2S effects in animals. In addition to facile genetic and genomic tools, using C. elegans provides the ability to precisely control both genotype and cellular H2S exposure. C. elegans grown in 50 ppm H2S are long‐lived and resistant to hypoxia‐induced disruption of protein homeostasis. The early transcriptional response to H2S requires the C. elegans orthologue of the hypoxia‐inducible transcription factor, hif‐1. HIF‐1 promotes survival in H2S, at least in part, by upregulating expression of sqrd‐1, which encodes the sulfide‐quinone oxidoreductase. SQRD‐1 catalyzes the first step in the mitochondrial oxidation of H2S. In an unbiased forward genetic screen, we found that expression of rhy‐1 suppresses lethality of hif‐1 mutant animals in 50 ppm H2S. RHY‐1 is an integral‐membrane ER protein with predicted acyltransferase (ACYL3) activity. The ACYL3 family is large and conserved across species from bacteria to primates, but the function of these enzymes is largely unstudied. RHY‐1 was first characterized as a negative regulator of HIF‐1, and our data indicate that it also promotes survival in H2S independently of HIF‐1. To understand RHY‐1 function we have used biotinylation by antibody recognition (BAR) to identify proteins that interact with RHY‐1. We show that RHY‐1 directly associates with CYSL proteins, which are orthologues of cystathionine β‐synthase. Mutations in cysl‐1 abrogate RHY‐1 function to regulate HIF‐1 and to promote survival independently of HIF‐1. We have also identified a novel methyltransferase, RIPS‐1, that is required for RHY‐1 to promote survival in H2S but which is dispensable for the proper regulation of HIF‐1. Together, our data show that RHY‐1 promotes survival in H2S through a novel mechanism which can bypass the only known catabolic pathway for H2S in vivo. Understanding the mechanism of RHY‐1 activity in H2S will begin to elucidate the relationship between H2S signaling and toxicity, and could lead to new therapeutic strategies to modulate endogenous H2S levels.

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