| Voltage-gated Ca2+ channels activate key physiological processes throughout the body, including contraction, secretion and expression. Given this rich biological context, it is no surprise that the mechanisms by which these channels are regulated attract intense investigation. Among the most intriguing forms of Ca2+ channel regulation is the feedback inactivation of L-type channels by intracellular Ca2+, acting via unconventional channel-calmodulin (CaM) interactions. In particular, previous studies hinted that Ca2+-free CaM (apoCaM) may “preassociate” with these channels, so as to enhance rapid detection of local Ca2+ . Despite the far-reaching consequences of this proposal, in vitro experiments testing for apoCaM preassociation provide conflicting results. The resulting obscurity surrounding preassociation has made it impossible to meaningfully engage crucial second order questions, such as how Ca 2+-CaM/channel interactions elicit inactivation. Here, we develop and employ novel strategies based on fluorescence resonance energy transfer (FRET) to directly probe associations between channels and CaM in single living cells, with green fluorescent protein (GFP) variants as fluorophore tags. Using this approach, we find clear evidence for significant association of apoCaM with intact L-type charnels. By scanning for associations between apoCaM and short channel fragments, we pinpoint two segments that collaboratively form a high-affinity apoCaM binding pocket. Surprisingly, one of these segments is an IQ domain that previously had been believed to only bind Ca2+ -CaM. Mutations in this domain are directly linked to disruption of apoCaM preassociation and, consequently, dramatic changes in channel function. Finally, we resolve substantial, Ca2+-induced conformational changes in CaM binding with short channel fragments, providing explicit evidence of a molecular trigger for Ca2+-dependent inactivation. Overall, these results significantly advance our molecular-level understanding of L-type channel regulation and underscore the potential of live-cell FRET for probing protein-protein interactions. |
