Ca2+ channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of Ca V 1-2 Ca 2+ channels are regulated by a single CaM constitutively complexed with channels. The Ca 2+-free form of CaM (apoCaM) is already pre-associated with the isoleucine-glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca 2+ binding to this resident CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel-CaM coordination could arise from the absence of Ca 2+ regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca 2+ feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca 2+ channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of Ca V 1.3 and Ca V 1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca 2+ feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca 2+ entry through channels is broadly significant for Ca 2+ homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca 2+ channels.
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