Voltage-gated calcium channels (Cavs) control movement, heartbeat, hormone secretion and brain activity. Malfunction of Cavs due to gene mutations or dysregulation causes cardiovascular, sensory and neurological diseases1. Cavs are targets of several blockbuster drugs1. What do Cavs look like? How do the different molecular components of Cavs intermingle? What makes a calcium channel a calcium channel? How do Cavs gate (i.e., open, close and inactivate)? Where and how do Cav drugs bind? How do disease-causing mutations alter Cav structure and function? A near-atomic resolution structure of a prototypical mammalian Cav obtained by Wu and colleagues and published recently in Nature2 (Figure 1) provides answers or clues to these interesting, important and long-pursued questions.

Figure 1
figure 1

Calcium channel structures — then and now. (A) Schematic of a Cav complex from a 2010 review3. This type of cartoon has been used to depict Cavs in the last 30 years. The inset shows a structure of a β-subunit bound with the AID, obtained in 2004 (PDB code: 1VYT). (B) Cryo-EM structure of the rabbit skeletal muscle Cav1.1 complex (PDB code: 5GJV) determined by Wu and colleagues in 20162. The different subunits are differentially colored, so are the four repeats, the AID, the III-IV linker and the proximal C-terminal domain (CTD) of the α1-subunit.

Cavs belong to the superfamily of voltage-gated ion channels. The so-called high-voltage activated Cavs are composed of a pore-forming α1-subunit and auxiliary α2δ-, β- and, in the case of the Cav1.1 complex elucidated by Wu et al., γ-subunits (Figure 1A). The α1-subunit, which contains four homologous but nonidentical repeats, dictates the major biophysical and pharmacological properties of Cavs, but the auxiliary subunits play key roles in regulating channel gating and trafficking1,3,4.

Crystal structures of Cav fragments in complex with calmodulin and of β-subunit in complex with its binding site in the α1-subunit (named the α-interacting domain, or AID) had been obtained some years ago3,5 (Figure 1A), and a crystal structure of CavAb, an engineered model bacterial Cav formed by four identical single-repeat subunits, has been determined recently6. However, before the recent game-changing breakthrough in single-particle cryo-electron microscopy (cryo-EM)7, high-resolution structures of mammalian Cavs seemed unachievable in the next 10 or even 20 years. This is not only because mammalian Cavs are composed of multiple subunits, but also because the multi-repeat α1-subunit is large (190-280 kDa) and contains numerous transmembrane helices and several long flexible regions, making them intractable by X-ray crystallography. Moreover, obtaining sufficient amount of pure, homogenous and fully-assembled Cav proteins suitable for X-ray crystallography was a daunting challenge.

Wu and colleagues solved the structure of the full Cav1.1 complex from the rabbit skeletal muscle by using single-particle cryo-EM, which circumvents many of the hurdles confronting X-ray crystallography. The authors used a clever strategy to obtain and purify the native Cav1.1 complex by replacing the endogenous β1a with a recombinant tagged β1a and taking advantage of the reversibility of the α1/β interaction3. A large dataset and sophisticated data processing aided the determination of the final 3.6 Å-resolution structure, improving upon an earlier lower-resolution structure obtained by the same group8.

The new structure provides unparalleled insights into the mechanisms of Cav assembly, ion permeation, gating and regulation. The structure confirms the pentameric architecture of Cav1.1 (Figure 1B). The four repeats of α1 enclose clockwise to form the asymmetric main body of the channel, with an ion conduction pore in the center. The intracellular end of S6, which forms the inner pore, is tightly closed. This, coupled with the 'up' position of the voltage-sensing S4 helix, suggests that the channel is in an inactivated state. The ion selectivity filter is formed by a ring of carboxylate side chains of the signature 'EEEE locus' and two rings of backbone carbonyl oxygens. This design of employing a combination of negatively charged side chains and main-chain carbonyls to coordinate ions is shared by other calcium-conducting channels, including CavAb6, TRPV17 and the type I ryanodine receptor9.

The Cav1.1 structure reveals how the auxiliary subunits interface with the α1-subunit (Figure 1B). The α2δ-subunit is shown to be cleaved, disulfide-bonded, glycosylated and anchored to the membrane, as previously described4, with the relevant site(s) identified. α2δ binds several extracellular loops of α1, the amino acid sequences of which diverge among different α1s; thus, the strength of the α1/α2δ interaction likely varies in different Cavs. A striking revelation is that α2δ protrudes far into the extracellular space, raising questions of whether it interacts with extracellular matrix proteins and/or other cell surface proteins and how such interactions alter Cav functions. The β-subunit binds the AID in the same manner as it does in isolated β/AID complexes. However, while the AID was previously envisioned to form a continuous α-helix with S6 of repeat I (IS6)3,5, the Cav1.1 structure shows that the AID helix is discontinuous with IS6 and runs nearly parallel to the membrane. This orientation places some β-subunit regions involved in gating modulation away from the transmembrane domains, suggesting that they may interact with α1 regions unresolved in the structure. The γ-subunit interacts primarily with the voltage-sensing domain of repeat IV, explaining its modulatory effect on gating3. Notably, the linker between repeats III and IV (the III-IV linker) of α1 interacts with the proximal C-terminal domain of α1, an interaction that may also modulate gating.

The determination of the first mammalian Cav structure marks a watershed in the studies of Cav structure-function and Cav-centric physiology, pharmacology and channelopathy. It will surely spur structural elucidation of other Cavs and investigation of the structural basis of Cav drug actions. Among these drugs, dihydropyridines (e.g., amlodipine), phenylalkylamines (e.g., verapamil) and benzothiazepines (e.g., diltiazem) are used to treat hypertension, angina pectoris and cardiac arrhythmias and target the α1-subunit of Cav1.2, whereas gabapentin and pregabalin are used to treat epilepsy and neuropathic pain and target the α2δ-subunit of Cavs in the nervous system1,4. The binding sites of dihydropyridines and phenylalkylamines have been identified recently in CavAb10, but whether these drugs bind in the same way in Cav1.2 remains to be determined. Based on the Cav1.1 structure, more accurate homology structure models of other Cavs can now be generated. By the same token, the locations of numerous disease-causing single amino acid missense mutations can now be mapped on the Cav1.1 structure and their effects on Cav structure/function can now be more precisely investigated. With the continuing rapid advance in structure biology, there is good reason to be optimistic that the structures of various macromolecular Cav complexes will be solved in the coming years, which will inform the molecular mechanisms of not only Cav functions but also excitation-contraction coupling, excitation-transcription coupling and excitation-secretion coupling.