Back to: Whitney Lab Home l Anderson Home

Regulation of Cellular Excitability

Peter A. V. Anderson, Ph.D.
Director of the Whitney Laboratory; Professor of Physiology and Functional Genomics and Neuroscience

Email: paa@whitney.ufl.edu

The focus of research in my lab is, in essence, to understand the basis and regulation of the excitability of cells. Our approach has been to capitalize on the experimental advantages presented by early eukaryotes, particularly cnidarians and flatworms. For example, in the case of the jellyfish Cyanea capillata, neurons in the motor nerve net (MNN) are large enough for intracellular recording, including voltage clamp analysis, allowing us to examine the physiology and pharmacology of the “early” nervous system. Moreover, synapes between MNN neurons are also readily accessible and amenable to intracellular recording, enabling chemical synaptic transmission to be examined in considerable detail.

At the same time, the phylogenetic separation between cnidarians and mammal provides a novel way of addressing the relationship between structure and function of specific molecules, including ion channels. In some instances, the function of ion channels and their subunits is highly conserved in both cnidarians and mammals. Under these circumstances, one can ignore structurally different parts of the molecule when trying to map function to structure. Alternatively, some functions, particularly pharmacology, are not conserved between cnidarians and mammals. In these case, structural differences between the cnidarian and mammalian proteins can be used to identify drug binding sites and other functions. In both instance, the information provided by the breadth of the phylogenetic comparison allows the “noise” normally present in comparisons of protein structure to be separated from the “signal”. Work in this broad area remains an on-going interest in my lab.

In recent years we have expanded our focus to include one of the most fascinating and arguably most complex cell in any eukaryote, the cnidarian cnidocyte, or sting cell.

ON-GOING PROJECTS

1. Identity of the neurotransmitter at MNN synapses.
Despite the amount of progress that has been made over the last 20 years in understanding the properties and capabilities of the cnidarian nervous system, one aspect of neuronal function that we still know relatively little about is the identity of neurotransmitters used by these animals, particularly the presence and role of the non-peptidergic neurotransmitters that are so prevalent in other nervous systems. While there is little doubt that neuropeptides are present, abundant and active in cnidarians, of all classes, a variety of reports suggest that more traditional neurotransmitters may also be present in cnidarians.

We have been taking advantage of the fact that MNN neurons and their synapses can be completely exposed to study of the action of a variety of neurotransmitter candidates, including those typically associated with fast synapses in higher animals, on synapses in the MNN. Only the amino acids taurine and beta-alanine produce physiologically responses consistent with those of the normal EPSP in these cells. Moreover, chemical analysis has revealed that both taurine and beta-alanine are present in the neurons and released by depolarization. These findings strongly suggest that either or both of these amino acids, or a closely related compound is the neurotransmitter at synapses between MNN neurons. We will be conducting additional studies aimed at confirming the role of these amino acids, or some analog thereof, at these synapses.

2. Regulation of Cnidocyte Discharge.
The cnidocytes of jellyfish and other cnidarians are extremely complex cells whose primary function, discharge, is regulated by a combination of very specific chemical and mechanical stimulation. We have been using a combination of electrophysiology, cell and molecular biology, including transcriptomic work, to understand the factors and cellular components that are involved in the regulation and mechanism of cnidocyte discharge.

Characterizing the transcriptome of cnidocytes.
We have been working on a DARPA-funded project that is exploring the possibility of using the cysts of cnidocytes as microscopic delivery systems for therapeutics and other agents. The ultimate goal of this work is to develop lines of cnidocytes that have been re-engineered so as to produce antidotes and other desirable compounds, rather than toxins, and to develop strategies for the controlled release of those agents into patients. We are focused on identifying the synthetic pathways used to make the toxins and package them into the cysts.

The approach we have taken to identify the synthetic pathways involved in toxin packaging is to sequence and annotate the transcriptome of cnidocytes. This approach would have the added benefit of providing useful information about a variety of other aspects of cnidocytes biology, including, potentially, the identification of all toxins, and details of the sensory pathways involved in the regulation of cnidocyte discharge.

Initial work focused on EST libraries created from whole tentacles and from various developmental stages of cnidocytes. Using a combination of EST and 454 sequencing, we have obtained a total of 8000 coding sequences. We plan to expand this and, ideally, fully characterize the transcriptome of these fascinating cells.

Ion channels in Cnidocytes.
We have cloned a variety of voltage-gated ion channels from cnidocytes from the Portugese Man-of-War, Physalia physalis. These included fragments of a Ca2+ channel alpha 1 subunit, a complete Ca2+ channel beta subunit (PpCavbeta) and a Shaker-like K+ channel (PpKv1). The functional properties of the latter two channel proteins were characterized electrophysiologically using heterologous expression.

While the presence of Ca2+ channel subunits in cnidocytes supports the model that discharge is a Ca2+-dependent exocytotic event, this finding must be interpreted cautiously. There is functional evidence that cnidocytes can form the pre-synaptic element of chemical synapses. Thus, Ca2+ channels are likely to be present in the cells, but localized near the basal membrane, where the synapses are located, rather than the apical membrane where discharge occurs. This distinction is important to our understanding of the Ca2+-dependency of cnidocyte discharge.

To distinguish between these two possibilities, we have developed an antibody against PpCavb and are screening cnidocytes from various species to identify its binding site and, therefore, the location of the Ca2+ channel beta subunit in cnidocytes.

Mechanosensory channels in cnidocytes.
Because cnidocytes are likely to be energetically expensive to make, on account of their complexity, and because they can only be used once, very elaborate mechanisms have evolved to ensure that they only discharge when there is a high likelihood of their successfully acquiring prey. One component of this regulatory pathway is the requirement that cnidocytes receive near simultaneous mechanical and chemical stimuli. Mechanical stimuli are transduced by the cnidocil and stereociliary complex at the apical end of the cnidocyte that projects into the surrounding sea water. We have been seeking to identify the molecular mechanisms underlying mechanotransduction by these receptors. Previous attempt to clone TRP channels, another class of putative mechanoreceptors channels, from cnidocytes had proved unsuccessful. In this project, we are attempted to clone mechanoreceptors of the ENaC family from cnidocytes.

Epithelial Sodium Channels (ENaC) are a family of transmembrane ion channels. They include mechanically-gated channels such as Mec-4 and Mec-10, which were cloned from C. elegans, and others that serve as receptors for pH, osmotic pressure, and neuropeptides. The Mec channels are the only proteins known to function as mechanoreceptors.

Conventional PCR-based cloning methods with degenerate primers derived from conserved regions of Mec channel proteins are being used to screen cDNA libraries obtained from intact tentacles and isolated, purified cnidocytes from Physalia. To date, an appropriately-sized fragement has been cloned and sequenced and found to have high sequence homology to ENaCs in general, and Mec-4 in particular. Two ENaC-homologous fragments have also been cloned from Cladonema.

Personnel

Peter A. V. Anderson, Professor
Christelle Bouchard, Assistant Research Scientist

Selected Publications

Anderson, Peter A. V. and Bouchard, C. (2009) The regulation of cnidocyte discharge. Toxicon, epub. ahead of press.

Oppegard, S. C., Anderson, Peter A. V. and Eddington, D.T. (2008) Cnidarian nematocysts as a functional component of a micro device. Bioinspiration and Biomimetics, submitted.

Seizer, H. M., Dorvel, B. R., Andersson, M., Fine, D., Price, R. B., Long, J. R., Dodabalpur, A., Koper, I., Knoll, W., Anderson, Peter A. V. and Duran, R.S. (2007) Functional ion channels in tethered bilayer membranes: implications for biosensor. Chembiochem. 8: 1246-1250.

Price, R. B. and Anderson, Peter A. V. (2006) Chemosensory pathways in the capitate tentacles of the hydroid Cladonema. Invert. Neuroscience,6, 23-32.

Bouchard, B., Price, R. B., Money penny, C. G., Thompson, L. F., Zillhardt, M., Stalheim, L. and Anderson, Peter A. V. (2006). Cloning and functional expression of voltage-gated ion channel subunits from cnidocytes of the Portuguese Man O'War, Physalia physalis. J. Exp. Biol. 209: 2979 - 2989.

Kohn, A.B., Roberts-Misterly, J.M., Anderson, P.A.V., Khan, N., and Greenberg, R.M. (2003). Specific residues in the Beta Interaction Domain of a schistosome Ca2+ channel [beta] subunit are key to its role in sensitivity to the antischistosomal drug praziquantel. Parasitology 127: 349-356.

Anderson, Peter A. V. (2004). Cnidarian Neurobiology: what does the future hold? Hydrobiologia, in press.
Anderson, Peter A. V., Roberts-Misterly, J. and Greenberg, R. M. (In press) The evolution of voltage-gated sodium channels: were algal toxins involved? Harmful Algae.

Kohn, A.B., Anderson, P.A.V., Roberts-Misterly, J.M., and Greenberg, R.M. (2002) Schistosome calcium channel ß subunits. Unusual modulatory effects and potential role in the action of the antischistosomal drug praziquantel. J. Biol. Chem. 276: 36873-36876.

Anderson, Peter A. V. and Greenberg, R.M. (2001). Phylogeny of Ion Channels: Clues to Structure and Function. Comp. Physiol. Biochem. 129B, 17-28.

Kohn, A.B., Lea, J.M., Roberts-Misterly, J.M., Anderson, P.A.V., and Greenberg, R.M. (2001). Structure of three high voltage-activated calcium channel alpha₁ subunits from Schistosoma mansoni. Parasitology 124: 489-497.

Kohn, A.B., Anderson, P.A.V., Roberts-Misterly, J.M., and Greenberg, R.M. (2001). Schistosome calcium channel ß subunits. Unusual modulatory effects and potential role in the action of the antischistosomal drug praziquantel. J. Biol. Chem. 276: 36873-36876.

Jeziorski, M.C., Greenberg, R.M. and Anderson, Peter A. V. (2000). The molecular biology of invertebrate voltage-gated Ca²⁺ channels, J. exp. Biol., 203: 841-856

Jeziorski, M.C., Greenberg, R.M. and Anderson, Peter A. V. (1999). Cloning and expression of a jellyfish calcium channel beta subunit reveal functional conservation of the alpha₁ - beta interaction. Receptors and Channels, 6: 375-386.

White, G.B, Pfahnl, A., Haddock, S., Lamers, S., Greenberg, R.M. and Anderson, Peter A.V. (1998). Structure of a Putative Sodium Channel from the Sea Anemone Aiptasia pallida. Invert. Neurosci., 3: 317-326.

Jeziorski, M.C., Greenberg, R.M., Clark, K.S. and Anderson, Peter A. V. (1998). Cloning and functional expression of a voltage-gated calcium channel alpha₁ subunit from jellyfish. J. Biol. Chem., 273: 22792-22799.

Blair, K.L. and Anderson, Peter A.V. (1996). Physiology and pharmacology of turbellarian neuromuscular systems. Parasitology 113: S73-S82.

Blair, K.L. and Anderson, Peter A.V. (1994). Physiology and pharmacology of muscle cells isolated from the flatworm Bdelloura candida. Parasitology 109: 325-335.

Anderson, Peter A.V., Holman, M. A. and Greenberg, R.M. (1993). Deduced amino acid structure of a putative sodium channel from the scyphozoan jellyfish Cyanea capillata. Proc. Natl. Acad. Sci. 90: 7419-7423.

Blair, K.L. and Anderson, Peter A.V. (1993). Properties of voltage-activated ionic currents in cells from the brains of the triclad flatworm Bdelloura candida. J. exp. Biol. 185: 267-286.

Anderson, Peter A.V., A. Moosler and C.J.P. Grimmelikhuijzen (1992). The distribution of AnthoRF-amide-like immunoreactivity in scyphomedusae. Cell Tiss Res. 267: 67-74.

Holman, M. and Anderson, Peter A.V. (1991). Voltage-activated ionic currents in myoepithelial cells from the sea anemone Calliactis tricolor. J. exp. Biol. 161: 333-346.

Anderson, Peter A.V. (1990). Evolution of the First Nervous Systems, Peter A. V. Anderson (Ed.). Plenum Press, New York. 423p.

Anderson, Peter A.V. and M. C. McKay (1987). The electrophysiology of cnidocytes. J. exp. Biol. 133: 215-230.

Anderson, Peter A.V. (1987). Properties and pharmacology of a TTX-insensitive Na⁺ current in neurones of the jellyfish Cyanea capillata. J. exp. Biol. 133: 231-248.