Identification of a voltage-gated proton channel gene in Karlodinium veneficum

1Department of Natural Sciences, University of Maryland Eastern Shore, Princess Anne MD 21853 2 University of Maryland Center for Environmental Science at the Institute of Marine and Environmental Technology, Baltimore MD 21202 Abstract #1042.3 Karlodinium veneficum, an ichthyotoxic dinoflagellate, is responsible for massive fish kills through the release of toxins (karlotoxins). The mechanism of release of the karlotoxin has not been elucidated. In bioluminescent dinoflagellates such as Noctiluca milaris and Lingulodinium polyhedra, voltage-gated proton channels serve as an unusual type of ion channel exhibiting several unique properties. It is known that voltage-gated proton channels form a low resistance pathway across the cell membrane that allows for the passage of particular ionic species in a controlled manner. Therefore, it was hypothesized that a voltagegated proton channel is present in K. veneficum and that permits the release of the karlotoxins. Identification of the gene suspected to code for this channel in the Karlodinium genome was accomplished using an EST library database created at University of Maryland Biotechnology Institute. Primers were designed using the Karlodinium EST Hv1 GI#06831923 sequence from the EST library and polymerase chain reaction was performed using reverse transcribed RNA from K. veneficum strain 1609. The 5’ end of the suspected proton channel gene in K. veneficum strain 1609 was identified and sequenced revealing the splice leader sequence at the 5’ end, four transmembrane domains and similarity with the EST identified in strain 2778. The 3’ end of the gene was not successfully sequenced. This gene will be ligated into a vector for expression which will permit for the study of its role in the release of karlotoxins. Supported by NIH T34GM008411