Title: Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.

Authors: M G Erickson, B A Alseikhan, B Z Peterson, D T Yue

Journal, date & volume: Neuron, 2001 Sep 27 , 31, 973-85

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/11580897

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 mutant CaM abolishes Ca(2+)-dependent modulation, hinting that Ca(2+)-free CaM may "preassociate" with these channels to enhance detection of local Ca(2+). Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca(2+)-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.