Toxic algal blooms pose a threat to fisheries and public health. A key issue for understanding the effects of toxic algae on aquatic ecosystems is how grazers evolve adaptations to algal toxins. Adaptation of copepods (the most abundant multicellular animals in the sea) to microalgae that produce potent neurotoxins (called saxitoxins, STX) has been previously demonstrated. STX blocks sodium (Na+) channels and interrupts nerve transmission signals. A novel mutation that is located in the inner portion of the Na+ channel, and leads to persistent electrical currents when the channel is inactivated (i.e., a leaky channel), has been identified. Leaky channels result in unintended nerve transmission signals and cell hypersensitivity; hence, they are considered to be very costly to individuals. However, it is hypothesized that in the presence of STX, the leaky channel mutation is advantageous because STX blockage of leaky channels reduces the likelihood of unintended nerve transmission signals and cell hypersensitivity. This project represents an interdisciplinary collaboration between labs at University of Connecticut and the University of Florida with expertise in molecular techniques, neurobiology, and zooplankton ecology and evolution. Expected results are: 1) Development of genetic markers for specifically detecting wild and mutant Na+ channels in individual copepods, 2) Neurophysiological characterization of the functional properties of the mutant Na+ channel and its responses to STX, 3) Quantification of the costs and benefits to individuals bearing the mutation responsible for the leaky Na+ channel. This research can potentially lead to the demonstration of a new molecular mechanism of neurotoxin adaptation, and inform a variety of fields of research from neurobiology to ecology and evolution. The broader impacts of this research are: 1) New key information for predicting whether grazers can control toxic blooms and toxin transfer to fish and shellfish. 2) Collaboration with professional educators to translate results from this study and the toxin adaptation literature into prepared curricular materials that meet national science standards. The material will be made widely available via a website and workshops presented at conference meetings of scientists and secondary educators. 3) Presentations in lecture series aimed at lay audiences. 4) Training of undergraduate and graduate students in an interdisciplinary environment.
This project sought to determine if the ability of some North Atlantic copepods to survive exposure to high concentrations of the neurotoxin, saxitoxin (STX), is the result of a natural mutation of their voltage-gated sodium channels. This possibility stems from the fact that some clams and salamanders are relatively insensitive to STX on account of structural changes in their sodium channels. The preliminary work that formed the basis for this study found that the only difference in the structure of sodium channels from STX-resistant and STX-sensitive copepods of the same species was a small three amino acid insertion and a single adjoining mutation in region immediately adjacent to the inactivation gate, which is located on the intracellular face of the protein. At first glance, this would not be the most obvious location for a structural change that might impact the action of STX since STX binds to specific amino acids on the extracellular face of the protein and is not membrane permeable. However, the same preliminary work provided evidence that sodium channels bearing these mutations did not inactivate completely and appeared to be functionally "leaky". This gave rise to the hypothesis that the ability of some copepods to resist the action of STX could be attributed to this leakiness. The goal of this project, therefore, was to conduct a rigorous electrophysiological study to fully characterize the properties of channels bearing the mutant, to test the hypothesis that the STX-resistance was attributable to leakiness and, if that were not the case, to try to identify other explanations for the phenomenon. The functional properties of cloned ion channels are typically determined by heterologous expression. Specifically, the messenger RNA (mRNA) for a particular channel is inserted into cells. Voltage clamp recording techniques are used to record currents flowing through the channels after the cell has translated the mRNA into a chain of amino acids, folded that protein into its correct configuration and inserted it into the cell’s membrane. Normally one would inject mRNA for the sodium channels from STX-resistant and STX-sensitive copepods into the cell of choice and record the resulting currents. However, invertebrate sodium channels are notoriously difficult to express and, direct expression of the copepod sodium channels was unsuccessful. In the case of the STX-resistant sodium channel from clams this problem was circumvented by mutating a small region of the Rat Brain IIA sodium channel, a well-studied channel that expressed routinely, to match the amino acid sequence of the clam sodium channel. We used the same approach. The functional properties of the Rat Brain IIA channel bearing the three amino acid insert and adjoining mutation (mutant channel) were compared with those of the native or Wild Type (WT) channel. We used two different expression systems; oocytes from the frog Xenopus and Human Embryonic Kidney (HEK) cells, each of which presents particular advantages. Our results confirmed the earlier finding that the mutant channels tended to be leakier than WT channels. Moreover, we found that mutant channels recover from inactivation faster than WT channels. Importantly, however, both WT and mutant channels were equally sensitive to STX – the dose/response curves for both the peak and the steady state (leaky) component of the currents were identical. No other differences between the channel types were found. Moreover, while we did find that Rat Brain IIA Na+ channels bearing the mutation were somewhat leaky, we are not convinced that copepod channels bearing the same mutation would also be leaky. The inactivation gate of a Na+ channel is thought to function by undergoing a conformational change that moves it into the pore where it binds to complementary residues and, thereby, occludes the pore. The three amino acid insert in the sodium channels of STX-resistant copepods will likely distort the inactivation gate such that it misaligns with the amino acids in the remainder of the Rat Brain IIA channel to which it would normally bind. This would explain the leakiness and the faster recovery from inactivation of the mutant channel. In copepods, the complementary residues to which the gate binds are presumably also positioned differently, thereby allowing correct binding and complete channel inactivation. Another possibility that cannot be tested without expressing the copepod channels directly is that the structural modifications inherent in Na+ channels from STX-resistant copepods may distort the overall channel structure to the point at which the residues on the extracellular face of the protein that normally bind to STX are slightly displaced, thereby reducing the efficacy of STX. The Broader Impacts of this study were achieved through a public lecture to an audience of approximately 150 people ranging in age from high school students through retirees. This lecture provided that audience with new insight into the role of neurotoxin in prey/predator relationships and generally broadened their understanding of biology and the marine sciences.
