One approach we currently use is to express cDNAs which encode calcium channels with fluorescent protein inserted at targeted sites (view animation), which permits the use of fluorescence resonance energy transfer (FRET), a technique that provides a sensitive measure of whether proteins are close (<10 nm) to one another. These experiments indicate that RyR1 may come into close apposition with two regions of the DHPR.
A second approach makes use of the expression of cDNAs which encode calcium channels in which the biotin acceptor domain (“BAD”) is attached to the targeted sites, resulting in the enzymatic addition of biotin at those sites. Subsequent exposure to fluorescent streptavidin reveals whether the accessibility of this large molecule to the site is influenced by the presence or absence of RyR1 (the results are in overall good agreement with the approach of using fluorescent proteins). In collaboration with Drs. Claudio Perez and Paul Allen, we are producing transgenic mice expressing DHPRs with site-directed biotinylation (Fig. 2). These mice allow us to purify DHPRs for single particle reconstruction, to carry out site-directed cross-linking, and to use gold particles attached to streptavidin to directly assess the orientation of specific DHPR sites with respect to RyR1.
The long term goal of our work is to determine regions of close proximity between the proteins (i.e., potential sites of interaction) within the TT/SR junction and also to determine whether those regions undergo dynamic changes during physiological function. The sorts of approaches we are developing may be of general use for analyzing the molecular machinery underlying important functions in many cell types.
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