| Abstract No.: | B-B2055 |
| Country: | Canada |
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| Title: | IDENTIFICATION AND CHARACTERIZATION OF TWO NOVEL INVERTEBRATE CHANNELS |
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| Authors/Affiliations: | 1 Adriano Senatore*; 1 David Spafford;
1 University of Waterloo, ON, Canada
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| Content: | Introduction: Heterologous expression of ion channels in a variety of cell culture systems has provided enormous insight into how these complex proteins contribute to the excitatory properties of neurons and other cell types. Integration of this type of data with work done in model organisms that have simple nervous systems in which functional units of neural circuitry can more easily be defined has put us at the forefront of understanding the mechanics of neural function. Our laboratory focuses on the invertebrate pond snail Lymnaea stagnalis, where we now have cloned four ion channel genes for characterization. Previously, Cav1 and Cav2 homologues (LCav1 and LCav2) had been cloned and characterized using cell culture and a variety of methods aimed at studying the function and localization of native ion channels in isolated neurons and whole animals. Here, we show that we have cloned two new Lymnaea ion channel genes, a T-type calcium channel and a NALCN cation leak channel homologue termed LNALCN.
Objectives: Our reverse genetics approach is aimed at characterizing both the electrophysiology of the channels in cell culture and in cultured neurons, and to determine their contribution to neural processes in Lymnaea. We hypothesize that the NALCN homologue is playing various roles in Lymnaea, including a role contributing to the cation leak seen in afterdischarge during egg-laying. The T type channels, which are active at rest, have small single-channel conductances and inactivate rapidly, will likely play a role in oscillatory networks in the snail such as the respiratory central pattern generator.
Results: Degenerate primers designed against the vertebrate T-type and NALCN channel coding sequences were used to screen Lymnaea cDNA libraries. The full coding sequence of both channels was determined by a combination of library screening, RT PCT, and RACE experiments. Several alternate splice variants were identified for both genes, including LNALCN, whose vertebrate counterpart has not yet been shown to undergo alternate splicing. The consensus sequences for both the Lymnaea T-type and LNALCN were cloned into pIRES2-EGFP bicistronic vector and are presently being studied in heterologous expression systems by electrophysiology and immunolabelling. Sothern blotting indicates that both channels are single copy genes, and northern blots and in situ hybridization are presently underway to determine what tissues/cell types express the two genes.
Conclusion: Understanding how our own nervous system works is arguably the most interesting subject of contemporary science. Unfortunately, due to the complexity of the system we have been forced to use a vast array of diverse approaches, some of which may have been rather subjective. With the advent of molecular biology and the revolution in scientific technology that is happening today, we are better equipped to objectively break down the problem and tease apart the modalities of neural function. Combining this with a reductionist approach using simpler model organisms has and will allow us to extrapolate to the mammalian nervous system to help us better understand how it all works. |
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