CAREER: Structural and Mechanistic Analysis of Potassium Channel Modulation by a Novel Activating Snake Toxin

Cuny College Of Staten Island, Staten Island NY

Investigators

Abstract

Potassium ion (K+) channels allow the rapid and specific flow of potassium ions across membranes in a regulated way, and are significant for many physiological functions in organisms from bacteria to humans. Opening of the channels and the resulting flow of K+ through the membrane is induced by a change in membrane voltage or by interaction with ligands. Because potassium channels are of such importance for normal physiological function, many organisms have evolved toxins that target channels, and these toxins have been used as important tools in the study of channel function and structure. Preliminary studies identified a toxin (Tx7335) from the eastern green mamba snake (Dendroaspis angusticeps) that increases frequency and duration of channel openings in the bacterial potassium channel KcsA. This is a very unusual function for a toxin that interacts directly with the potassium-conducting domain of the channel, as most such toxins act as blockers of K+ currents. The functional effect of Tx7335 therefore represents a new mechanism of K+-channel regulation. The main research objective of this project is to understand the basis for the functional effect of Tx7335, and in particular the details of the interaction between channel and toxin and the structural changes that toxin binding induces in the channel. In order to probe the surface of the channel that interacts with the toxin, individual amino acid residues on the external surface of KcsA that may interact with the toxin will be mutated to a different amino acid. If the original amino acid is indeed important in the interaction, then the expectation is that the mutation will lead to a different response of the mutant channel to the toxin. The response will be tested by measuring potassium currents flowing through all mutant channels in artificial bilayers with and without added toxin. A rough mapping of the toxin binding site will be possible in this way. Mutations will also be made on the toxin to identify residues on the surface of the toxin that interact with the channel. Solution nuclear magnetic resonance spectroscopy (NMR) experiments will be used to obtain higher-resolution structural information of the channel-toxin interactions. This technique allows the characterization of the binding interface and the detection of structural changes upon toxin binding to be performed with atomic resolution. These experiments will eventually allow resolving the three-dimensional toxin-channel complex structure. This knowledge will advance understanding of K+ channel function and regulation, with relevance for a large number of biological processes in animals that are dependent on influx of potassium ions. In addition, this understanding will also be relevant to plant physiology, where highly homologous potassium channels are involved in potassium uptake and transport as well as in the opening and closing of stomata by regulating the turgor pressure of the surrounding guard cells. Lastly, ion channel toxins selective for insect channels may also be very useful as insecticides with reduced negative environmental impact, and better understanding of toxin-channel interactions will be beneficial in guiding the development of such pest control approaches in agriculture. Broader Impact The project will provide an opportunity for the integration of research in education and will contribute to the training of the next generation of students in Staten Island and the greater New York City area with a special emphasis on students who are historically underrepresented in science. Undergraduate and high-school students participants will be recruited from the College of Staten Island, from LaGuardia Community College (a minority-serving institution), and from high-need local Staten Island high schools. Students will be closely mentored to promote development of their scientific and critical thinking skills and to increase their preparation to succeed in science. An outreach program through the Discovery Institute at the College of Staten Island will involve developing and disseminating teaching modules based on the proposed research suitable for implementation at the high-school level. These programs will be created by interested teachers in collaboration with the PI and Discovery Institute experts, and will be based on analysis of structural data pertaining to the project using molecular visualization software. Students will suggest mutations that would affect toxin binding and upload these to the project web site. Submissions will be functionally tested by the research staff and the final experiments will be streamed back to the high school and archived as video podcasts on the project server. The implementation of these modules will help to create excitement for science and engage students in hypothesis-driven research through media that the students are already familiar with (computer 3D software, webcasts and online video). Lastly, the curriculum for the undergraduate biochemistry laboratory course that the PI teaches at the College of Staten Island will also be redesigned as a research-based course drawing from elements of the research project. This exposure to actual research in a mandatory lab course will enrich student's understanding of the nature of science and the design of research. In summary, the research will serve as a vehicle to increase scientific interest, literacy and fluency among involved undergraduate students as well as local high-school teachers and students.