A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are shortlived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.natural products | secondary metabolism | biosynthesis | RiPP | cyanobactin T here are two fundamentally different types of metabolic pathways in living systems. The first are aimed to generate one or a few discrete chemicals; these comprise the majority of pathways. The second have evolved to produce large numbers of different metabolites (1, 2). Although perhaps fewer in number, this second class, which we will call "diversity generating" (DG), may be responsible for the majority of small molecules in living systems. A key difference is that the compounds produced in the latter generally have a more limited phylogenetic distribution.The metabolic pathways first elucidated were for the synthesis of essential metabolites found in all cells, such as amino acids, purines, or pyrimidines. These conventional metabolic pathways typically comprise multiple metabolic steps, with the intermediates generated in each step converted only to the final product of the pathway. DG pathways, however, do not yield a single final product. Each enzyme in a DG pathway has relaxed substrate specificity and is able to handle a variety of compounds, carrying out the same chemical transformation on different substrates.Previously, an evolutionary framework was developed to explain why some biosynthetic pathways produce many compounds (3-5). In this study we provide the first integrated overview, to our knowledge, of the multiple metabolic steps that comprise a DG biosynthetic pathway. We have uncovered striking differences in how this pathway differs from the canonical features of conventional pathways. Our results provide an initial framework for understanding how DG pathways are designed and how key features of such pathways diverge from the textbook model.We specifically examine the tru and related pat cyanobactin pathways (1, 6). These ribosomally synthesized and posttranslationally modified (RiPP) secondary metabolite pathways were identified in cyanobacterial symbionts of coral reef animals. Their expression required transfer to a model host, Escherichia coli (Fig.

Chronic pain affects the life of millions of people. Current treatments have deleterious side effects. We have advanced a strategy for targeting protein interactions which regulate the N-type voltage-gated calcium (CaV2.2) channel as an alternative to direct channel block. Peptides uncoupling CaV2.2 interactions with the axonal collapsin response mediator protein 2 (CRMP2) were antinociceptive without effects on memory, depression, and reward/addiction. A search for small molecules that could recapitulate uncoupling of the CaV2.2–CRMP2 interaction identified (S)-lacosamide [(S)-LCM], the inactive enantiomer of the Food and Drug Administration–approved antiepileptic drug (R)-lacosamide [(R)-LCM, Vimpat]. We show that (S)-LCM, but not (R)-LCM, inhibits CRMP2 phosphorylation by cyclin dependent kinase 5, a step necessary for driving CaV2.2 activity, in sensory neurons. (S)-lacosamide inhibited depolarization-induced Ca2+ influx with a low micromolar IC50. Voltage-clamp electrophysiology experiments demonstrated a commensurate reduction in Ca2+ currents in sensory neurons after an acute application of (S)-LCM. Using constellation pharmacology, a recently described high content phenotypic screening platform for functional fingerprinting of neurons that uses subtype-selective pharmacological agents to elucidate cell-specific combinations (constellations) of key signaling proteins that define specific cell types, we investigated if (S)-LCM preferentially acts on certain types of neurons. (S)-lacosamide decreased the dorsal root ganglion neurons responding to mustard oil, and increased the number of cells responding to menthol. Finally, (S)-LCM reversed thermal hypersensitivity and mechanical allodynia in a model of postoperative pain, and 2 models of neuropathic pain. Thus, using (S)-LCM to inhibit CRMP2 phosphorylation is a novel and efficient strategy to treat pain, which works by targeting specific sensory neuron populations.

A cone snail venom peptide, μO §-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligandbinding site. μO §-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ SSG ). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na V 1.2. A mutant channel of rNa V 1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10 3 -fold less sensitive to GVIIJ SSG than was wild-type rNa V 1.2. In contrast, although rNa V 1.5 was >10 4 -fold less sensitive to GVIIJ SSG than Na V 1.2, an rNa V 1.5 mutant with a cysteine in the homologous location, rNa V 1.5[L869C], was >10 3 -fold more sensitive than wildtype rNa V 1.5. The susceptibility of rNa V 1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa V β2 or rNa V β4, but not that of rNa V β1 or rNa V β3, protected rNa V 1.1 to -1.7 (excluding Na V 1.5) against block by GVIIJ SSG . Thus, GVIIJrelated peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na V β-and Na V α-subunits are present in native neurons.oltage-gated sodium channels (VGSCs) are responsible for the upstroke of action potentials in excitable tissues. Each VGSC is composed of a pore-and voltage sensor-bearing α-subunit and one or more auxiliary β-subunits. Mammals have nine α-subunit isoforms (Na V 1.1 to -1.9) and four β-subunit isoforms (Na V β1 to -β4) (1). An Na V 1 has about 2,000-aa residues arranged in four homologous domains, where each domain has six transmembrane spanning segments with an extracellular "pore" loop between segments 5 and 6 (1, 2); furthermore, each Na V 1 has about a dozen extracellular cysteine residues, all located in or near the pore loops. For the most part, not much is known about these cysteines (including whether they are disulfide bonded).Na V β-subunits can affect the function and cellular localization of Na V 1s (1, 3-5). Each Na V β-subunit has some 200-aa residues and consists of a single transmembrane segment with a large extracellular domain and a smaller intracellular domain (1). Na V β2-and Na V β4-subunits, unlike Na V β1-and Na V β3-subunits, are disulfide bonded to α-subunits (1, 6). A given neuron can have multiple isoforms of these subunits whose identities are challenging to appraise pharmacologically (7).Toxins that target VGSCs have been invaluable for probing the structure and function of these channels. Venoms are a rich source of such toxins. For example, in Conus snails, four families of neuroactive peptides have been characterized that target VGSCs:...

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology." calcium imaging | conotoxin | sensory neuron | neuronal subtype E ach type of neuron is programmed to have distinctive signaltransduction pathways and electrical properties. At any given anatomical locus in the nervous system, functionally diverse neuronal subtypes are present, which diverge in the specific mix of ion-channel and receptor isoforms expressed. Recently, we demonstrated that different neuronal subclasses exhibit distinct functional phenotypes in response to a panel of pharmacological challenges. We organized such pharmacological challenges into a profiling platform for discriminating between the various neuronal subclasses present in a heterogeneous population of dissociated neurons (1).In the present study, we demonstrate the feasibility of targeting a minor fraction of dorsal root ganglion (DRG) neurons for comprehensive functional profiling, to the point where the probable physiological role of the neurons characterized can be inferred and a targeted physiological intervention becomes feasible. A key strength of this approach is that it enables the identification and functional characterization of neuronal subclasses that represent only a miniscule fraction of a diverse cell population. A key cellular...

We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor-agonist challenges) and compounds that affect voltage-gated ion channels (membrane-potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor-agonist and membrane-potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system. sensory neuron | neuronal subpopulation | conotoxin | conopeptide | Fura-2