Technical Reference #232

232.

Preassociation of Calmodulin with Voltage-Gated Ca2 Channels Revealed by FRET in Single Living Cells Michael G. Erickson; Badr A. Alseikhan; Blaise A. Peterson; David T. Yue, Johns Hopkins University School of Medicine, Neuron, 31(232), (2001)
Link To Paper

Abstract:
Among the most intriguing forms of Ca(2+) channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca(2+) acting via unconventional channel-calmodulin (CaM) interactions. In particular overexpressing Ca(2+)-insensitive mutan

Materials & Methods:
Three-cube FRET is by no means the only way to mea- of three filter-cube determinations performed on single sure steady-state CFP/YFP FRET but many of the exist- cells with a standard epifluorescence microscope. ing methods (Selvin 1995) are not easily adapted into Moreover three-cube FRET explicitly establishes and microscope-based assays of fluorescent proteins ex- implements key simplifications for optical dissection pressed in single living cells. Measurements of fluores- permitted by the CFP/YFP pair (Figure 2A). These simplicence lifetime (Harpur et al. 2001) for example require fications enable FR to be computed entirely with experispecialized light sources and detectors capable of mentally determined factors that completely correct for quantifying the nanosecond lifetimes of GFP variants. optical peculiarities of the microscope system. In addi- The methods that may be most readily tailored for stan- tion the focus on sensitized YFP emission favors sensidard fluorescence microscopes entail measurements of tive detection of FRET because YFP is much brighter relative emission intensities of the CFP and/or YFP using than CFP (Patterson et al. 2001). A distinct advantage of commonly available optical filters to select for specific three-cube FRET is that it explicitly accounts for partial excitation and emission wavelengths. Here we review binding of donor/acceptor molecules thereby permitselected examples of this group of FRET detection ting extraction of relative affinity parameters (Figure 5). methods and highlight some of the special advantages To our knowledge the ratiometric FCET and FRETN of our three-cube FRET algorithm for studying channel/ approaches have not incorporated such a capability. CaM preassociation. Finally three-cube FRET can be generalized to other The simplest approach is to compute the ratio of the novel fluorophore pairs such as GFP/DsRed or CFP/ YFP/CFP emission peaks at 535 nm to 480 nm (Miya- DsRed (Moon et al. 2001). waki et al. 1997). Though this method is ideal for as- A remaining strategy accounting for variable fluorosessing dynamic FRET it is poorly suited for detecting phore expression requires selective destruction of dosteady- state FRET in single cells because variable ex-

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HEK-293