Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.
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.
Erickson
M G
MG
Department of Biomedical Engineering, Calcium Signals Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Alseikhan
B A
BA
Peterson
B Z
BZ
Yue
D T
DT
D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000737:chemistry / Q000378:metabolism*; D020908:Calcium Channels, R-Type / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004735:Energy Transfer; D005246:Feedback; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D015640:Ion Channel Gating; D008164:Luminescent Proteins / Q000032:analysis; D046911:Macromolecular Substances; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D014162:Transfection
D002118:Calcium; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D020908:Calcium Channels, R-Type; D002147:Calmodulin; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins