Genetics - Theses

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Start date: 2003 × End date: 2003 × Author: Vargas-Delgado, Ernesto. ×

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    Functional analysis of conserved domains in the Drosophila TRPL channel
    Vargas-Delgado, Ernesto. (University of Melbourne, 2003)
    The Drosophila Transient Receptor Potential (TRP) and Transient Receptor Potential Like (TRPL) proteins form non selective cation channels whose activity depolarize the photoreceptor neurons in response to light. The initial steps that trigger the photoreceptor response are well characterized. However, the molecular mechanisms that directly activate and regulate the channels remain unknown. The elucidation of critical domains and their contribution to channel activation might constitute a starting point to understand the activating mechanism of TRPL and other TRP channels. The function of conserved regions amongst TRP channels was explored by creating mutations in the TRPL channel (truncations and point mutations) in three regions of the protein with high conservation through human and Drosophila TRP channels. The first conserved domains studied were the Ankyrin Repeats (AR) motifs that are present in the Amino-terminal region of the TRPC and TRPV subfamilies of TRP channels. These motifs were deleted in the TRPL channel, expressed in HEK cells and studied by patch-clamp whole cell recordings. Two truncated channels lacking all or only the 3rd and 4th AR showed activation by GTP-y-S included in the patch pipette, indicating that these conserved motifs do not play a key role in TRPL channel activation. Within the transmembrane domains region, the loop linking the S4 and S5 transmembrane domains, which shows significant degree of conservation, was studied. The substitution of Leucine 542 and Serine 545 for Alanines (mutant LS) did not affect TRPL activation, whereas changing Glycine 540 and Glutamine 543 for Alanines (mutant GO) eliminated the channel response to GTP-?-S. The possible role of this region was also investigated by dialysis of a peptide derived from this S4-S5 loop into HEK cells expressing wild type TRPL. This treatment elicited currents in cells expressing TRPL as well as in cells expressing the human homologue TRPC6. In overlay assays this peptide bound a Drosophila head protein of approximately 200 KDa. In conjunction these results indicate that the region is necessary for normal channel activation and that it probably regulates TRPL channel activity by interacting with other proteins. The C-terminal region was the third region investigated. The deletion of aminoacids 682-1025 (mutant ?C1), located immediately C-terminal to the last transmembrane domain, abolished TRPL channel responses to both GTP-?-S and Linolenic acid (LNA). The deletion of the highly conserved region 682-698, which is present in all TRPC channels, significantly reduced the response to GTP-?-S and prevented LNA activation of the channel. These results provide evidence of the regions within the channel that are required for its normal activation by GTP-?-S or by lipids. Although, the actual role that these identified regions is playing in channel functioning can not be established, different possible scenarios are discussed.