| Calmodulin (CaM) in complex with Ca2+ channels constitutes a prototype for Ca2+ sensors that are intimately co-localized with Ca2+ sources. The C-lobe of CaM senses local, large Ca 2+ oscillations due to Ca2+ influx from the host channel, and the N-lobe senses global, albeit diminutive Ca2+ changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca2+ sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and experimentally validate this proposal with methodologies enabling millisecond control of Ca 2+ oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca2+ release from CaM combined with greater affinity of the channel for Ca2+-free versus Ca 2+-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca2+ sources.;Beyond the initiation of CaM-mediated channel regulation, we further investigate the end-stage transduction of two key processes---voltage-dependent inactivation (VDI), and CaM-mediated Ca2+-dependent Inactivation (CDI). Previous dogma suggests that both VDI and CDI share a common 'hinge-lid' mechanism, wherein a cytosolic channel domain binds to and occludes the channel pore. Here, we utilize an S6 mutagenesis screen to validate an alternate view, in which CDI and VDI are actually quite different. CDI proceeds via an allosteric modulation of channel gates, quite different from the hinge-lid hypothesis. VDI is consistent with a modified version of the hinge-lid mechanism, which we call the 'hinge-lid/shield' mechanism. These findings are not only of mechanistic interest, but provide a potentially rich backdrop for understanding the many heritable diseases that involve mutations in S6 domains.;Finally, looking ahead, future tests of Ca2+ decoding mechanisms will likely require FRET-based readouts of conformational changes in the channel complex. Though ratiometric quantification of CFP/YFP FRET seems well suited to this problem, this approach has met with limited success on more advanced imaging platforms, such as confocal microscopes. Thus, we characterize sources of optical variability (unique to the confocal context), and devise practical remedies which permit remarkably consistent quantification of subcellular FRET in live cells. |
