Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells

Michael G. Erickson, Badr A. Alseikhan, Blaise Z. Peterson, David T. Yue

Research output: Contribution to journalArticle

Abstract

Among the most intriguing forms of Ca2+ channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca2+, acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca2+-insensitive mutant CaM abolishes Ca2+-dependent modulation, hinting that Ca2+-free CaM may "preassociate" with these channels to enhance detection of local Ca2+. 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 Ca2+-independent associations between CaM and the pore-forming α1 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.

Original languageEnglish (US)
Pages (from-to)973-985
Number of pages13
JournalNeuron
Volume31
Issue number6
DOIs
StatePublished - Sep 27 2001

ASJC Scopus subject areas

  • Neuroscience(all)

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