20.109:TR Red Mod4 research proposal

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TR Red Mod4 Research Proposal: Tracking conformational changes in sodium channels with fluorescent tagging

Overview

We want to create a better way to measure the conformational state of an ion channel. We propose tagging an ion channel protein with GFP such that it will only fluoresce when the channel is in a one conformational state. Possible ideas for creating this conformational differentiability are circularly-permutated GFP and FRET pair geometry.

Background

Ion channels and other membrane transport proteins play a crucial role in a cell's interaction with its environment. A lot of interest has been placed in the study and tracking of ion channels, particularly in neurons, because of this. While methods exist to track the conformation of an individual channel (patch clamping), there is currently no way to look at the conformation of different channels on a systems level. Previous research has been successful in tagging K+ ion channels while maintaining the function of the channel (Kupper, 1998); however, the addition of a conformation dependent tag has not yet been done. Having a conformation-dependent fluorescent channel will be useful as it will allow better tracking of opening and closing of ion channels throughout the entire cell, or potentially even the entire tissue.

Statement of the Problem

It is currently difficult to determine whether ion channels in a cell are open or closed. Patch clamping is able to measure this for one ion channel at a time, but it would be ideal to measure this noninvasively for every ion channel of a specific type in a cell.

Project Details and Methods

Predicted Outcome

Resources Required

References

Biskup, C., Zimmer, T., & Benndorf, K. (2004). FRET between cardiac na+ channel subunits measured with a confocal microscope and a streak camera. Nature biotechnology, 22(2), 220-224.

Chanda, B., & Bezanilla, F. (2002). Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. The Journal of General Physiology, 120(5), 629-645.

Fry, M., Porter, D. M., & Maue, R. A. (2003). Adenoviral-mediated expression of functional na+ channel ?1 subunits tagged with a yellow fluorescent protein. Journal of neuroscience research, 74(5), 794-800.

Jurgen, K. (1998). Functional expression of GFP-tagged Kv1.3 and Kv1.4 channels in HEK 293 cells. European Journal of Neuroscience, 10(12), 3908-3912.

Levitan, E. S. (1999). Tagging potassium ion channels with green fluorescent protein to study mobility and interactions with other proteins. In P. Michael Conn (Ed.), Methods in enzymology (pp. 47-58)Academic Press.

Massensini, A. R., Suckling, J., Brammer, M. J., Moraes-Santos, T., Gomez, M. V., & Romano-Silva, M. A. (2002). Tracking sodium channels in live cells: Confocal imaging using fluorescently labeled toxins. Journal of Neuroscience Methods, 116(2), 189-196.

Riely, B. K., Lougnon, G., Ane, J. M., & Cook, D. R. (2007). The symbiotic ion channel homolog DMI1 is localized in the nuclear membrane of medicago truncatula roots. The Plant Journal : for cell and molecular biology, 49(2), 208-216.

Staruschenko, A., Medina, J. L., Patel, P., Shapiro, M. S., Booth, R. E., & Stockand, J. D. (2004). Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial na+ channel. Journal of Biological Chemistry, 279(26), 27729-27734.

Szanda, G., Koncz, P., Varnai, P., & Spat, A. (2006). Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains. Cell calcium, 40(5-6), 527-537.

Treves, S., Pouliquin, P., Moccagatta, L., & Zorzato, F. (2002). Functional properties of EGFP-tagged skeletal muscle calcium-release channel (ryanodine receptor) expressed in COS-7 cells: Sensitivity to caffeine and 4-chloro-m-cresol. Cell Calcium, 31(1), 1-12.

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