- Email: [email protected]
Visualizing Individual Sodium Channels on the Move
Chemistry & Biology
Previews can reduce the toxic side effects of nifurtimox, it will be a tremendous boon to Chagas disease treatment and another example of the unexpected benefits of phenotype-based screening and target identification.
REFERENCES Castro, J.A., de Mecca, M.M., and Bartel, L.C. (2006). Hum. Exp. Toxicol. 25, 471–479. Hall, B.S., Bot, C., and Wilkinson, S.R. (2011). J. Biol. Chem. 286, 13088–13095.
Mayr, L.M., and Bojanic, D. (2009). Curr. Opin. Pharmacol. 9, 580–588.
Zhou, L., Ishizaki, H., Spitzer, M., Taylor, K.L., Temperley, N.D., Johnson, S.L., Brear, P., Gautier, P., Zeng, Z., Micthell, A., et al. (2012). Chem. Biol. 19, this issue, 883–892.
Visualizing Individual Sodium Channels on the Move Stefan H. Heinemann1,* 1Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, D-07745 Jena, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2012.07.002
Visualization of voltage-gated sodium channels at work is an important requirement for the understanding of rapid electrical signaling in nerve cells. In this issue of Chemistry & Biology, Ondrus and colleagues have mastered this challenge by chemical synthesis of a fluorescent antagonist and by monitoring single sodium channels in living cells with unprecedented optical resolution. Voltage-gated sodium channels (NaV channels [NaVs]) are large transmembrane proteins that mediate an influx of Na+ into neurons and muscle cells and thereby generate action potentials. Importance of NaVs for the rapid electrical signaling is manifested by various neuronal and muscle diseases that originate from NaVs dysfunction, and the specific pharmacological interference with NaVs holds promise to cure some of these neuro or muscular disorders and to combat various forms of pain. The molecular function and the physiological roles of NaVs are tightly linked to where in the organism and, even more precisely, where in a neuron they reside. Moreover, since nine genes code for NaV channels in humans (Goldin, 2002; Figure 1A), individual NaV subtypes may occur at distinct sites to serve specific cellular functions. With some exceptions, however, functional properties of NaVs, such as the membrane voltage and kinetics of channel opening and closing, are only subtly different, thus seriously limiting the ability to identify specific NaVs expressed in the central nervous system. Classification of select NaV types based on their kinetic properties only is an intricate matter; in particular, as functional evaluation would also require perfectly
controlled electrical access to the cellular substructures of interest, this is de facto impossible. The discovery of the small guanidiniumderived molecules tetrodotoxin (TTX) and saxitoxin (STX) that specifically bind to and inhibit NaVs was therefore a significant step forward in the early 1960s (e.g., Narahashi et al., 1967). TTX has been instrumental for classifying NaVs in TTX-sensitive and TTX-resistant channels, and it is now well established that a single residue in the pore region of NaVs largely determines the variations in their sensitivity toward TTX (Figure 1A; Noda et al., 1989; Terlau et al., 1991; Backx et al., 1992). STX shares the binding site with TTX in the outer vestibule of the channel’s pore and blocks the flow of Na+ more potently than TTX (half-maximal inhibitory concentration of about 2 nM). Channel-toxin interaction follows a 1:1 stoichiometry, and the average dwell time of an STX molecule at the channels is on the order of 1 min. In nature, STX is produced by marine dinoflagellates, which, when accumulated by filter-feeding shellfish, can give rise to paralytic shellfish poisoning. While de novo synthesis of the complex STX molecule was a limiting factor for a long time, radioactively labeled biosynthetic STX was developed as a potent and specific
790 Chemistry & Biology 19, July 27, 2012 ª2012 Elsevier Ltd All rights reserved
marker of NaVs and used to detect and count channel molecules in various preparations (Ritchie et al., 1976). Radioactive STX, however, is not suited to gain information on the cellular or even subcellular distribution and dynamics of NaVs, whereas specific labeling of NaVs with a fluorescent probe holds promise to give a better insight into such molecular mechanisms of rapid cellular signaling. However, NaVs are sparse, labeling by means of antibodies is often complicated, and tagging the channels with genetically engineered green fluorescent protein is not applicable to native preparations. In addition, optical visualization of NaVs at their sites of physiological operation faces another problem, because these channels are expressed in very tiny neuronal substructures, such as neuritic spines and boutons, with dimensions below the diffraction limit of light microscopy, which, according to Abbe’s law, typically is 200 nm. In this issue of Chemistry & Biology, Ondrus et al. (2012) provide a breakthrough by combining two approaches: chemical synthesis of STX and its fluorescent derivatives and application of superresolution fluorescent microscopy to image NaVs in living cells at a single-molecule level. They linked the fluorescent
Chemistry & Biology
Previews
Figure 1. Fluorescent STX Targets a Subset of Voltage-Gated Sodium Channels (A) Dendrogram of human voltage-gated sodium channels (NaV1.1–NaV1.9) with their major site of expression and an indication of an amino acid residue in the pore loop of channel domain I that largely determines the sensitivity of the channels toward TTX and STX. Bars indicate approximate IC50 values for TTX. CNS, central nervous system; PNS, peripheral nervous system; DRG, dorsal root ganglia. (B) Chemical structure of STX coupled via nitrogen 21 (arrow) and an NH3 linker (black) to the fluorophore Cy-5 (red) (Ondrus et al., 2012). (C) Cartoon illustrating how STX-Cy5 transiently labels NaVs to allow for dynamic super-resolution imaging with minimal bleaching. Future NaV labels may be suited for discriminating between different channel isoforms (light and dark green).
cyanine dye Cy-5, which is characterized by red fluorescence, high quantum yield, and photostability, to the N21 position of the STX molecule (Figure 1B; STX-Cy5). When attached to that position, the extra fluorophore lowers the toxin’s ability to bind to NaVs by about a factor of 20 but still can be considered a potent and selective NaV label. Using single-particle detection methods beyond the optical diffraction limit, the authors could monitor the dynamics of NaVs in neuritic spines at real time and thus opened the door to very detailed studies on the contribution of sodium channel trafficking to neuronal plasticity on a subcellular level. STX-Cy5 therefore appears to be a very powerful novel tool for studying the implication of voltage-gated sodium channels in neuronal signaling (Figure 1C). The advantages of fluorescently labeled STX variants, relatively small size and water-solubility, come at the expense of moderate NaV subtype specificity (see Figure 1A) and a relatively large impact of the fluorophore on the binding affinity of the scaffold toxin molecule. This is exemplified in the STX-dichlorofluorescin variant, which blocks NaVs about 50 times
less potently than STX (Ondrus et al., 2012). Using peptide-based toxins such as scorpion a- or b-toxins of about 60–70 amino acid residues, the impact of a fluorophore is expected to be smaller (Massensini et al., 2002). Moreover, such peptides may add specificity to distinguish NaV1.2, NaV1.6, and NaV1.7 channels (Leipold et al., 2004), and with the appropriate fluorescent label, they are likely to also be suited for superresolution microscopy. The fluorescent labeling of m-conotoxins might be even more powerful. Such peptides of about 20 residues from the venoms of marine cone snails are small enough to allow straightforward synthetic production; they share the binding site at the channel with TTX and STX but may provide better discrimination of various forms of STXsensitive neuronal NaVs. However, all such approaches have in common that the selective label also affects the function of the channel under consideration. One future direction of development, therefore, could be to devise channelselective dyes that bind to but do not impede channel function to let them shine bright and sharp while they are
performing tasks.
their
electrical
signaling
REFERENCES Backx, P.H., Yue, D.T., Lawrence, J.H., Marban, E., and Tomaselli, G.F. (1992). Science 257, 248–251. Goldin, A.L. (2002). J. Exp. Biol. 205, 575–584. Leipold, E., Lu, S.Q., Gordon, D., Hansel, A., and Heinemann, S.H. (2004). Mol. Pharmacol. 65, 685–691. Massensini, A.R., Suckling, J., Brammer, M.J., Moraes-Santos, T., Gomez, M.V., and RomanoSilva, M.A. (2002). J. Neurosci. Meth. 116, 189–196. Narahashi, T., Haas, H.G., and Therrien, E.F. (1967). Science 157, 1441–1442. Noda, M., Suzuki, H., Numa, S., and Stu¨hmer, W. (1989). FEBS Lett. 259, 213–216. Ondrus, A.E., Lee, H.D., Iwanaga, S., Parsons, W.H., Andresen, B.M., Moerner, W.E., and Du Bois, J. (2012). Chem. Biol. 19, this issue, 902–912. Ritchie, J.M., Rogart, R.B., and Strichartz, G.R. (1976). J. Physiol. 261, 477–494. Terlau, H., Heinemann, S.H., Stu¨hmer, W., Pusch, M., Conti, F., Imoto, K., and Numa, S. (1991). FEBS Lett. 293, 93–96.
Chemistry & Biology 19, July 27, 2012 ª2012 Elsevier Ltd All rights reserved 791
Previews can reduce the toxic side effects of nifurtimox, it will be a tremendous boon to Chagas disease treatment and another example of the unexpected benefits of phenotype-based screening and target identification.
REFERENCES Castro, J.A., de Mecca, M.M., and Bartel, L.C. (2006). Hum. Exp. Toxicol. 25, 471–479. Hall, B.S., Bot, C., and Wilkinson, S.R. (2011). J. Biol. Chem. 286, 13088–13095.
Mayr, L.M., and Bojanic, D. (2009). Curr. Opin. Pharmacol. 9, 580–588.
Zhou, L., Ishizaki, H., Spitzer, M., Taylor, K.L., Temperley, N.D., Johnson, S.L., Brear, P., Gautier, P., Zeng, Z., Micthell, A., et al. (2012). Chem. Biol. 19, this issue, 883–892.
Visualizing Individual Sodium Channels on the Move Stefan H. Heinemann1,* 1Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena and Jena University Hospital, D-07745 Jena, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.chembiol.2012.07.002
Visualization of voltage-gated sodium channels at work is an important requirement for the understanding of rapid electrical signaling in nerve cells. In this issue of Chemistry & Biology, Ondrus and colleagues have mastered this challenge by chemical synthesis of a fluorescent antagonist and by monitoring single sodium channels in living cells with unprecedented optical resolution. Voltage-gated sodium channels (NaV channels [NaVs]) are large transmembrane proteins that mediate an influx of Na+ into neurons and muscle cells and thereby generate action potentials. Importance of NaVs for the rapid electrical signaling is manifested by various neuronal and muscle diseases that originate from NaVs dysfunction, and the specific pharmacological interference with NaVs holds promise to cure some of these neuro or muscular disorders and to combat various forms of pain. The molecular function and the physiological roles of NaVs are tightly linked to where in the organism and, even more precisely, where in a neuron they reside. Moreover, since nine genes code for NaV channels in humans (Goldin, 2002; Figure 1A), individual NaV subtypes may occur at distinct sites to serve specific cellular functions. With some exceptions, however, functional properties of NaVs, such as the membrane voltage and kinetics of channel opening and closing, are only subtly different, thus seriously limiting the ability to identify specific NaVs expressed in the central nervous system. Classification of select NaV types based on their kinetic properties only is an intricate matter; in particular, as functional evaluation would also require perfectly
controlled electrical access to the cellular substructures of interest, this is de facto impossible. The discovery of the small guanidiniumderived molecules tetrodotoxin (TTX) and saxitoxin (STX) that specifically bind to and inhibit NaVs was therefore a significant step forward in the early 1960s (e.g., Narahashi et al., 1967). TTX has been instrumental for classifying NaVs in TTX-sensitive and TTX-resistant channels, and it is now well established that a single residue in the pore region of NaVs largely determines the variations in their sensitivity toward TTX (Figure 1A; Noda et al., 1989; Terlau et al., 1991; Backx et al., 1992). STX shares the binding site with TTX in the outer vestibule of the channel’s pore and blocks the flow of Na+ more potently than TTX (half-maximal inhibitory concentration of about 2 nM). Channel-toxin interaction follows a 1:1 stoichiometry, and the average dwell time of an STX molecule at the channels is on the order of 1 min. In nature, STX is produced by marine dinoflagellates, which, when accumulated by filter-feeding shellfish, can give rise to paralytic shellfish poisoning. While de novo synthesis of the complex STX molecule was a limiting factor for a long time, radioactively labeled biosynthetic STX was developed as a potent and specific
790 Chemistry & Biology 19, July 27, 2012 ª2012 Elsevier Ltd All rights reserved
marker of NaVs and used to detect and count channel molecules in various preparations (Ritchie et al., 1976). Radioactive STX, however, is not suited to gain information on the cellular or even subcellular distribution and dynamics of NaVs, whereas specific labeling of NaVs with a fluorescent probe holds promise to give a better insight into such molecular mechanisms of rapid cellular signaling. However, NaVs are sparse, labeling by means of antibodies is often complicated, and tagging the channels with genetically engineered green fluorescent protein is not applicable to native preparations. In addition, optical visualization of NaVs at their sites of physiological operation faces another problem, because these channels are expressed in very tiny neuronal substructures, such as neuritic spines and boutons, with dimensions below the diffraction limit of light microscopy, which, according to Abbe’s law, typically is 200 nm. In this issue of Chemistry & Biology, Ondrus et al. (2012) provide a breakthrough by combining two approaches: chemical synthesis of STX and its fluorescent derivatives and application of superresolution fluorescent microscopy to image NaVs in living cells at a single-molecule level. They linked the fluorescent
Chemistry & Biology
Previews
Figure 1. Fluorescent STX Targets a Subset of Voltage-Gated Sodium Channels (A) Dendrogram of human voltage-gated sodium channels (NaV1.1–NaV1.9) with their major site of expression and an indication of an amino acid residue in the pore loop of channel domain I that largely determines the sensitivity of the channels toward TTX and STX. Bars indicate approximate IC50 values for TTX. CNS, central nervous system; PNS, peripheral nervous system; DRG, dorsal root ganglia. (B) Chemical structure of STX coupled via nitrogen 21 (arrow) and an NH3 linker (black) to the fluorophore Cy-5 (red) (Ondrus et al., 2012). (C) Cartoon illustrating how STX-Cy5 transiently labels NaVs to allow for dynamic super-resolution imaging with minimal bleaching. Future NaV labels may be suited for discriminating between different channel isoforms (light and dark green).
cyanine dye Cy-5, which is characterized by red fluorescence, high quantum yield, and photostability, to the N21 position of the STX molecule (Figure 1B; STX-Cy5). When attached to that position, the extra fluorophore lowers the toxin’s ability to bind to NaVs by about a factor of 20 but still can be considered a potent and selective NaV label. Using single-particle detection methods beyond the optical diffraction limit, the authors could monitor the dynamics of NaVs in neuritic spines at real time and thus opened the door to very detailed studies on the contribution of sodium channel trafficking to neuronal plasticity on a subcellular level. STX-Cy5 therefore appears to be a very powerful novel tool for studying the implication of voltage-gated sodium channels in neuronal signaling (Figure 1C). The advantages of fluorescently labeled STX variants, relatively small size and water-solubility, come at the expense of moderate NaV subtype specificity (see Figure 1A) and a relatively large impact of the fluorophore on the binding affinity of the scaffold toxin molecule. This is exemplified in the STX-dichlorofluorescin variant, which blocks NaVs about 50 times
less potently than STX (Ondrus et al., 2012). Using peptide-based toxins such as scorpion a- or b-toxins of about 60–70 amino acid residues, the impact of a fluorophore is expected to be smaller (Massensini et al., 2002). Moreover, such peptides may add specificity to distinguish NaV1.2, NaV1.6, and NaV1.7 channels (Leipold et al., 2004), and with the appropriate fluorescent label, they are likely to also be suited for superresolution microscopy. The fluorescent labeling of m-conotoxins might be even more powerful. Such peptides of about 20 residues from the venoms of marine cone snails are small enough to allow straightforward synthetic production; they share the binding site at the channel with TTX and STX but may provide better discrimination of various forms of STXsensitive neuronal NaVs. However, all such approaches have in common that the selective label also affects the function of the channel under consideration. One future direction of development, therefore, could be to devise channelselective dyes that bind to but do not impede channel function to let them shine bright and sharp while they are
performing tasks.
their
electrical
signaling
REFERENCES Backx, P.H., Yue, D.T., Lawrence, J.H., Marban, E., and Tomaselli, G.F. (1992). Science 257, 248–251. Goldin, A.L. (2002). J. Exp. Biol. 205, 575–584. Leipold, E., Lu, S.Q., Gordon, D., Hansel, A., and Heinemann, S.H. (2004). Mol. Pharmacol. 65, 685–691. Massensini, A.R., Suckling, J., Brammer, M.J., Moraes-Santos, T., Gomez, M.V., and RomanoSilva, M.A. (2002). J. Neurosci. Meth. 116, 189–196. Narahashi, T., Haas, H.G., and Therrien, E.F. (1967). Science 157, 1441–1442. Noda, M., Suzuki, H., Numa, S., and Stu¨hmer, W. (1989). FEBS Lett. 259, 213–216. Ondrus, A.E., Lee, H.D., Iwanaga, S., Parsons, W.H., Andresen, B.M., Moerner, W.E., and Du Bois, J. (2012). Chem. Biol. 19, this issue, 902–912. Ritchie, J.M., Rogart, R.B., and Strichartz, G.R. (1976). J. Physiol. 261, 477–494. Terlau, H., Heinemann, S.H., Stu¨hmer, W., Pusch, M., Conti, F., Imoto, K., and Numa, S. (1991). FEBS Lett. 293, 93–96.
Chemistry & Biology 19, July 27, 2012 ª2012 Elsevier Ltd All rights reserved 791