Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells. Journal   Neuron. Citation   Neuron. 31(6):973-85 Publication date   2001 Sep 27 Authors   Erickson MG Alseikhan BA Peterson BZ Yue DT Investigators   Blaise Z. Peterson MeSH headings   Calcium Calcium Channels, L-Type Calcium Channels, N-Type Calcium Channels, R-Type Calmodulin Spectrometry, Fluorescence MeSH qualifiers   metabolism methods 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. Medline ID   21465220