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that SyrB2 might catalyze promiscuous reactions if native halides were substituted with other ions. Reconstitution of the SyrB2 system, including the SyrB1 carrier protein loaded with a model unactivated aliphatic substrate, l-2-aminobutyrate, showed that SyrB2 can indeed install alternative anions in particular, N3

– and NO2

– in place of a halide. Although yields using wild-type SyrB2 were very low (~4% and 8%, respectively), formation of alkylazides and nitroalkanes in this fashion is unprecedented in biology. Addition of a glycine mutation designed to create more space for the larger N3

– and NO2

– ions respectively increased activity to ~20% and 50% and suggests that further improvements may be achievable through evolution. Fortuitously, introduction of this glycine also reduced native halogenation activity and inhibition by contaminating chloride, a feature that may mimic respecialization toward promiscuous enzyme function in nature. Equilibrium binding experiments showed that a variety of additional anions, including CN–, OCN–, HS– and HCO2

–, are also capable of accessing the halide-binding site. Although no C-H activation products were observed with these reagents, other coupling reactions may be achievable through engineering or by exploring alternative halogenase scaffolds.

Despite the novelty of this enzymatic C-N bond formation reaction, there remain severe limitations, including low product yield, that mitigate the current usefulness of this approach for chemical synthesis. For example, all of the halogenases in this family require substrates covalently attached to accessory carrier proteins (for example, SyrB1), which substantially limits substrate scope. Nevertheless, two additional SyrB1 substrates, threonine and norvaline, also show detectable levels of nitration, indicating that SyrB2 can access alternative substrates if given the chance. These challenges offer exciting opportunities for continued exploration.

The power of this work highlights how detailed mechanistic insight, informed by chemical ingenuity, can provide access to new modes of biocatalysis. In this simplified approach, new chemistry may be achieved by merely interrogating natural enzymes, particularly metalloenzymes, with reagents that are rare or may not exist in the environment. This is in line with recent discoveries by Arnold and associates6–8 showing that cytochrome P450s can access metallocarbenoid and nitrenoid insertion reactions when provided with non-natural diazoacetate and sulfonazide reagents. Alternative strategies for engineering new enzyme activity through the immobilization of non-natural

metallocenters have gained traction in recent years but have been limited to highly specialized protein scaffolds9,10. Approaches that access new reactivity through the promiscuous use of synthetic reagents may markedly expand our ability to engineer non-natural catalysis by making use of the vast diversity of enzymes that already exist in nature. ■

Eric M. Brustad is at the Department of Chemistry, Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. e-mail: brustad@unc.edu

Published online 26 January 2014 doi:10.1038/nchembio.1457

References1. Bornscheuer, U.T. et al. Nature 485, 185–194 (2012). 2. Chen, M.S. & White, M.C. Science 318, 783–787 (2007). 3. Matthews, M.L. et al. Nat. Chem. Biol. doi:10.1038/nchembio.1438

(26 January 2014).4. Khersonsky, O. & Tawfik, D.S. Annu. Rev. Biochem. 79,

471–505 (2010). 5. Matthews, M.L. et al. Proc. Natl. Acad. Sci. USA 106,

17723–17728 (2009). 6. Coelho, P.S. et al. Science 339, 307–310 (2013). 7. McIntosh, J.A. et al. Angew. Chem. Int. Ed. Engl. 52, 9309–9312

(2013). 8. Wang, Z.J. et al. Chem. Sci. 5, 598–601 (2014). 9. Wilson, M.E. & Whitesides, G.M. J. Am. Chem. Soc. 100,

306–307 (1978). 10. Hyster, T.K. et al. Science 338, 500–503 (2012).

Competing financial interestsThe author declares no competing financial interests.

TRP CHANNELS

Pain enters through the side doorTRPM3 can permeate ions through two distinct pores—the central pore and a likely alternative ‘omega pore’. Entry of ions through the alternative pore of TRPM3 contributes to pain generation, making it an attractive target for the design of new analgesics.

Emily R Liman

The prevailing dogma and an abundance of evidence tells us that voltage-gated ion channels contain a

single central permeation pathway formed at the juncture of the four channel subunits (or pseudosubunits). This conclusion, supported by crystallographic data from channels as divergent as voltage-gated K+ channels1 and ligand- and heat-gated transient receptor potential (TRP) channels2, has important implications for the development of pharmacological agents that block ion entry. Now, Vriens et al.3 challenge this ‘central pore dogma’ by showing that the ion channel TRPM3 can conduct ions through an

alternative pathway opened upon exposure to specific chemical signals.

TRPM3 is a member of the family of TRP ion channels, a family that contains a number of ion channels involved in sensory signaling. TRPM3, which was first cloned in 2003, remained an orphan channel, without a ligand or validated function4, until it was discovered that the neuroactive steroid pregnelone sulfate (PS) was a potent activator of the channel5. Although the conditions under which elevated PS levels gate the channel are not known, the availability of a validated agonist has allowed detailed study of

TRPM3. Most surprisingly, TRPM3 was found to be expressed in nociceptors, where, along with the better-studied TRP channel family members TRPV1 and TRPA1, it contributes to the generation of pain6. Accordingly, intraplantar injection of PS elicits nocifensive behavior not observed in TRPM3 knockout mice. Indeed, hypersensitivity in response to inflammatory mediators is reduced in TRPM3 knockout animals, to a similar extent as observed in TRPV1 knockout animals.

But here is where the story gets a little bit strange. In search of TRPM3

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modulators, Vriens et al.3 noticed that clotrimazole (Clt), a drug used to treat yeast infections, caused PS-activated TRPM3 currents, which are ordinarily outwardly rectifying, to acquire an inwardly rectifying component at negative voltages (Fig. 1). This observation defies explanation in terms of channel gating; instead, through an extensive series of biophysical experiments, Vriens et al.3 show that it reflects the permeation of cations through two separate pores in the channel. One is the well-described central pore. The other, alternative, pore may be akin to the ‘omega pore’ or ‘gating pore’ observed in some mutant voltage-gated K+ and Na+ channels7–9. The segment of the channel comprising the omega pore ordinarily functions as the passageway for the charged residues of the voltage sensor (S4) region, but mutation of the first of these charged residues in voltage-gated K+ and Na+ channels can open the way for positively charged ions as large as guanidinium7–9. This pathway is unmasked in several diseases, including hypokalemic periodic paralysis, where mutation of a

sodium channel generates an omega pore and produces a leak current at rest10.

The authors’ evidence that TRPM3 has a naturally occurring alternative pore that can be opened with the right combination of agonists is exhaustive. It includes the observations that the Clt- plus PS-induced inward current is not sensitive to agents (La3+) or mutations that block the central pore and that the current persists under conditions that inactivate the central pore. The S4 region of TRPM3 is uncharged and therefore could function as an omega pore and carry the inwardly rectifying current. Indeed, introduction of a charged residue in the first position of the S4 region (W982R) specifically disrupts the Clt- plus PS-induced inward current, which is consistent with this possibility.

The presence of an alternative pore in TRPM3 might seem like a biophysical oddity observed only under very contrived conditions in the laboratory. But Vriens et al.3 provide evidence that it occurs in vivo and can contribute to the genesis of pain signals. This is substantiated by the observation that intraplantar injection of mice with Clt alone does not produce

nocifensive responses but does potentiate responses to injection of PS in a TRPM3-dependent manner. Could Clt act on another target in vivo? This has not yet been resolved beyond doubt and probably will not be until tools to selectively block the alternative pore become available. Moreover, as Clt is clearly not the endogenous agonist for this channel, one is left wondering whether an inflammatory mediator has the same pore-opening property, and if so, which one? Finally, molecular determinants for the alternative pore in TRPM3 have not been well defined; this may require structural information, something that is within reach given the recent determination of the structure of TRPV1 (ref. 2).

TRPM3 represents one of the first examples of a naturally occurring channel with what appears to be an omega pore, and this alone will generate excitement among biophysicists. Stimulated by these results and with additional scrutiny, researchers will undoubtedly find alternative pores in other ion channels that contain uncharged S4 regions. Identifying the mechanisms by which specific ligands open the alternative pore in TRPM3 or other channels is a challenge for the future. Additionally, the discovery that the alternative pore of TRPM3 contributes to the genesis of pain signals makes it an attractive target for new analgesics. In the future, we can look forward to the development of the alternative or omega pore blockers to complement the central pore blockers already available for many ion channels. ■

Emily R. Liman is in the Section of Neurobiology, Department of Biological Sciences, University of Southern California, Los Angeles, Los Angeles, California, USA. e-mail: liman@usc.edu

References1. Doyle, D.A. et al. Science 280, 69–77 (1998). 2. Liao, M., Cao, E., Julius, D. & Cheng, Y. Nature 504, 107–112

(2013). 3. Vriens, J. et al. Nat. Chem. Biol. 10, 188–195 (2014).4. Nilius, B. & Voets, T. Nat. Cell Biol. 10, 1383–1384 (2008). 5. Wagner, T.F. et al. Nat. Cell Biol. 10, 1421–1430 (2008). 6. Vriens, J. et al. Neuron 70, 482–494 (2011). 7. Starace, D.M. & Bezanilla, F. Nature 427, 548–553 (2004). 8. Tombola, F., Pathak, M.M. & Isacoff, E.Y. Neuron 45,

379–388 (2005). 9. Sokolov, S., Scheuer, T. & Catterall, W.A. Neuron 47,

183–189 (2005). 10. Sokolov, S., Scheuer, T. & Catterall, W.A. Nature 446,

76–78 (2007).

Competing financial interests The author declares no competing financial interests.

Figure 1 | TRPM3 channels can permeate ions through an alternative pore to stimulate pain behaviors. (a) Agonist-activated TRPM3 currents are outwardly rectifying but develop an inwardly rectifying component in response to a combination of the agonist, a neurosteroid (PS) and the channel modulator clotrimazole (Clt). (b) The inwardly rectifying component is not carried by the central pore and is likely to be carried by an alternative pore formed in part by the S4 domain. (c) Nociceptors express TRPM3 and respond vigorously to concurrent application of PS and Clt. (d) Mice respond with nocifensive behaviors to intraplantar injection of PS and Clt, and this response is TRPM3 dependent.

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