In the Sherlock Holmes tale The Adventure of the Dancing Men, the detective runs a heart-pounding race to try to save his client’s life. The thumping of the sleuth’s heart — a literary example of the ‘fight-or-flight’ effect1 — reflects the changes that occur when the entry of calcium ions into the heart rises2. Writing in Nature, Liu et al.3 provide a solution to the long-standing riddle of how this occurs, through deductions worthy of Sherlock Holmes.

Read the paper: Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics

Some aspects of how calcium enters the heart during a fight-or-flight response are known. The process is mediated by the hormone adrenaline acting on β-adrenergic receptors — proteins that reside in the surface membrane of heart cells called cardiomyocytes. Receptor activation leads to an increase in the opening of what is called an L-type voltage-gated calcium channel. This occurs through a mechanism that involves the molecule cyclic AMP (cAMP)4,5 and an enzyme called protein kinase A (PKA) that requires cAMP for its function6. Similar types of PKA-mediated processes are found in other contexts. For example, some neurons use cAMP and PKA to enhance calcium entry through L-type calcium channels7.

Exactly how the stimulation of β-adrenergic receptors modulates calcium-ion influx has been debated since the 1970s. Researchers have uncovered tantalizing clues to the identity of the target molecule that PKA modifies by the addition of a phosphate group (phosphorylation). However, proposals for specific candidate targets, phosphorylation sites8–10 and modulatory mechanisms have been repeatedly called into question by further tests, including some in painstakingly constructed mouse models11–13. Liu et-al. use a powerful technique called proximity proteomics14 to implicate a previously under-appreciated suspect15 and to establish its role.

L-type voltage-gated channels provide a route by which calcium ions enter cardiomyocytes to help trigger a heartbeat. If channel opening is boosted, this results in a stronger and faster heartbeat. Previous investigations of how PKA might modulate channel opening focused mainly on amino-acid residues in the channel that occur in structural motifs possibly phosphorylated by PKA. But when Liu and colleagues performed a tour-de-force experiment in mice in which the channel was engineered so that all candidate phosphorylation sites were converted to an amino acid (alanine) that can’t be phosphorylated, and when this channel was studied alone, PKA-mediated enhancement of L-type channels nevertheless persisted. The authors therefore looked elsewhere for the elusive mediator of PKA’s ability to regulate the fight-or-flight effect.

The tornadoes of sudden cardiac arrest

Reasoning that some unknown factor must come into close proximity to the calcium channel during this regulatory process, the authors conducted a systematic search. Using proximity proteomics, Liu and colleagues engineered channel subunits to contain an enzyme that adds a tag called biotin to any protein within a radius of approximately 20 nanometres15. Tagged proteins were then identified by mass spectrometry. Hundreds of proteins in proximity to the calcium channel were analysed, and the authors found that the protein Rad was enriched in the channel microenvironment under resting conditions, but was noticeably depleted during stimulation of the β-adrenergic receptor. This dovetailed with an earlier clue — Rad is known to inhibit L-type voltage-gated calcium channels15, and, in mice, deletion of the gene that encodes Rad mimics the effect of β-adrenergic stimulation and eliminates further adrenaline-mediated enhancement of the activity of L-type channels16.

Liu et al. investigated whether PKA could prevent Rad-mediated channel inhibition. The authors tested whether phosphorylation of amino-acid residues on Rad would enable it to move away from the vicinity of the calcium channel. They narrowed the candidate residues down to four serines (in some experiments, just two), which, if replaced by alanine, abolished PKA-mediated regulation of calcium entry.

The calcium channel’s β-subunit was the prime suspect as the target of Rad inhibition. Ablation of the interaction between the calcium channel’s α 1C -subunits and its β-subunits fully eliminates PKA-mediated modulation of channel activity17. Indeed, the authors’ measurements, using a technique called fluorescence resonance energy transfer, showed that the interaction between Rad and the calcium-channel β-subunit was inhibited by PKA phosphorylation of the key serines in Rad that the authors had identified. Further tightening the noose around Rad’s metaphorical neck, electrical recordings demonstrated that all of the biophysical fingerprints of modulation by β-adrenergic signalling — such as the activity of previously inactive calcium channels and a shift in the voltage dependence of their activation18 — were prevented by eliminating Rad phosphorylation.

The results make a compelling case for the following scenario (Fig. 1). Adrenaline binds and activates the β-adrenergic receptor. This, in turn, results in the activation of an enzyme that produces cAMP, which activates PKA. PKA phosphorylates Rad and causes it to leave the vicinity of the calcium channel, thereby preventing it from inhibiting the channel.

Figure 1 | Modulation of the cardiac calcium channel. In heart cells called cardiomyocytes, the activity of calcium-ion channels increases during what is called the fight-or-flight response. Activation of the enzyme protein kinase A (PKA) is required for this process, and, in mouse studies, Liu et al.3 reveal that its elusive target is the protein Rad. a, In the absence of a fight-or-flight response, the β-adrenergic receptor is not stimulated and PKA is inactive. Rad binds to a subunit of the calcium channel (beige; only the α 1C - and β-channel subunits are shown) and calcium-ion (Ca2+) entry into cardiomyocytes is low. b, During the fight-or-flight response, the hormone adrenaline activates the β-adrenergic receptor. This leads to the production of cyclic AMP (cAMP) molecules, which activate PKA. Activated PKA adds a phosphate group (P) to Rad, causing Rad to dissociate from the channel and enabling channel activity to increase. This elevation of Ca2+ in the cytoplasm boosts the heartbeat.

The study puts Rad and other members of this family of proteins front and centre as players in calcium-channel modulation. Is Rad the entire missing chapter in the story of PKA’s role in the heart, given Liu and colleagues’ compelling arguments that other potential PKA targets are unnecessary? Sceptics will want further in vivo evidence from a type of mouse model termed a knock-in — animals whose original Rad sequence is replaced either with a version in which Rad’s own PKA-phosphorylation sites are mutated or with a version in which the part of Rad needed for the interaction with the β-subunit is eliminated — to see whether any PKA-mediated modulation of the calcium channel still occurs. Hints of differences between channel regulation in the embryonic and adult heart13 also warrant further study.

Might cardiac regulation by Rad be of clinical value? Heart failure in humans is associated with loss of regulation of calcium channels by β-adrenergic receptors. Rad levels fall during heart failure19, perhaps providing a temporary increase in the strength of heart contraction16. However, this would also reduce the heart’s ability to further increase its strength20, what is known as its functional reserve, which would be a severe price for a person’s heart to pay.

There will undoubtedly be debate about how PKA modulation of calcium channels operates in neurons, such as in PKA-responsive CA1 pyramidal cells in which Rad is essentially absent. In those neurons, the mutation of a particular serine (serine 1928) to alanine in the L-type channel eliminates channel modulation and L-type channel-dependent strengthening of inter-neuronal (synaptic) connections7. Here, PKA might be phosphorylating the calcium channel, after all.

Organ-specific pathways for regulation would make functional sense. Rad can completely inhibit calcium-channel activity, and so modifying such inhibition would give heart cells a wide range of regulatory capability18, suitable for a brief flight-or-fight response. Perhaps other cell types needing a more sustained but subtler boost to their calcium-channel activity might operate better without Rad-mediated regulation and rely instead on milder, more direct modulation of a subunit of the calcium channel.

Liu and colleagues have set a high bar for future detective work on cellular signalling in the heart. Their work shows the power of a systematic round-up of suspects and relentless interrogation of their roles.