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

@article{citeulike:775637, 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.}, address = {Department of Biomedical Engineering, Calcium Signals Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.}, author = {Erickson, M. G. and Alseikhan, B. A. and Peterson, B. Z. and Yue, D. T.}, citeulike-article-id = {775637}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/11580897}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=11580897}, day = {27}, issn = {0896-6273}, journal = {Neuron}, keywords = {algorithm, fret, stoichiometry}, month = {September}, number = {6}, pages = {973--985}, posted-at = {2006-07-27 11:16:09}, priority = {2}, title = {Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11580897}, volume = {31}, year = {2001} }

TY - JOUR ID - citeulike:775637 L3 - citeulike-article-id:775637 N2 - 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. IS - 6 JF - Neuron SN - 0896-6273 AD - Department of Biomedical Engineering, Calcium Signals Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. EP - 985 TI - Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells. VL - 31 SP - 973 KW - algorithm KW - fret KW - stoichiometry AU - Erickson, MG AU - Alseikhan, BA AU - Peterson, BZ AU - Yue, DT PY - 2001/09/27/ UR - http://view.ncbi.nlm.nih.gov/pubmed/11580897 ER -