Counting CaMs at calcium channels

How many calmodulin (CaM) molecules does it take to turn off a calcium channel? It sounds like the start to a bad joke, but Mori and colleagues have tackled this question using channels fused to CaM molecules — and the answer, they say, is just one.

CaM interacts with calcium channels to carry out two functions: it mediates calcium-dependent inactivation of the channels, and it transduces the signal from calcium entry through the channels into various intracellular signalling pathways. Mori et al. fused CaM molecules to L-type calcium channels to investigate the stoichiometry of these processes.

In the absence of calcium, CaM molecules pre-associate with L-type calcium channels. When calcium subsequently enters through the channel and interacts with CaM, the channel is inactivated. The authors used polyglycine linkers to fuse an inactive, mutant CaM (CaM_MUT) to each L-type calcium channel, on the principle that the CaM_MUT would act as a dominant-negative and prevent wild-type CaM from associating with the channel. Channel–CaM_MUT chimaeras did not undergo calcium-dependent inactivation, but channels fused to a wild-type CaM did, indicating that the fused CaM is important for inactivation.

To test whether other CaM molecules were needed for inactivation to occur, Mori et al. overexpressed free CaM_MUT along with L-type calcium channels. Unfused channels showed no inactivation in these circumstances, because the CaM_MUT was occluding the pre-association site and preventing wild-type CaM from inactivating the channels. However, channels that were fused to a wild-type CaM showed normal inactivation, demonstrating that a single CaM molecule is both necessary and sufficient for calcium-dependent inactivation.

In another part of the study, the authors investigated the local concentration of endogenous CaM around channels. They took advantage of the variable lengths of polyglycine linkers. When short linkers are used, CaM_MUT completely occludes the pre-association sites and calcium-dependent inactivation is blocked. With longer linkers, however, the local concentration of CaM_MUT drops, allowing endogenous CaM to access the pre-association sites and to mediate calcium-dependent inactivation. This effect can be used to estimate the local concentration of endogenous CaM near the channels. The authors calculated that the level of local endogenous CaM at the pre-association locus was around 2.5 mM — much higher than in the cell as a whole.

Such local enrichment of CaM near L-type calcium channels should allow the calcium influx through one channel to activate many CaM molecules, so that either many signalling pathways can be activated or a single pathway can be amplified.

These insights into the stoichiometry of channel regulation and signalling by CaM should help to advance our understanding of the mechanisms by which CaM carries out these functions. The approach of linking molecules to investigate their interactions should also be a valuable technique for researchers in other fields, and will no doubt produce many more fascinating results.