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A Crash Course in Calcium Channels Gerald W. Zamponi* Hotchkiss Brain Institute and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary T2N 1N4, Canada ABSTRACT: Much progress has been made in understanding the molecular physiology and pharmacology of calcium channels. Recently, there have been tremendous advances in learning about calcium channel structure and function through crystallography and cryo-electron microscopy studies. Here, I will give an overview of our knowledge about calcium channels, and highlight two recent studies that give important insights into calcium channel structure. KEYWORDS: Calcium channels, physiology, pharmacology
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function. Structure−function studies based on the generation of chimeric channels and site directed mutagenesis focused on understanding features such a voltage-dependent inactivation, interactions with calcium channel agonists and antagonists, pinpointing sites of second messenger regulation, and identifying interaction sites for regulatory proteins that are involved in channel trafficking and various physiological functions.1 Hence, despite the lack of a channel structure, much progress in understanding how these channels behave has occurred. Another important advance that arose from the cloning of the channels was their link to genetic mutations associated with a wide range of disorders.2,3 Mutations or sequence deletions in various calcium channel subunits have been reported in different inbred mouse lines with severe phenotypes such as cerebellar ataxia and absence seizures. More importantly, in humans, disease causing mutations in different Cavα1 subunits have been identified in patients with congenital stationary night blindness (Cav1.4), spinocerebellar ataxia type 6 (Cav2.1), familial hemiplegic migraine (Cav2.1), hypokalemic periodic paralysis (Cav1.1), Timothy syndrome (Cav1.2), congenital deafness (Cav1.3), and absence epilepsy (Cav3.2).2,3 These findings underscore the fundamental importance for voltage gated calcium channels for human physiology and pathophysiology. They are also mirrored in phenotypes observed with mouse lines deficient for specific calcium channel subunits. Calcium channel dysregulation can also occur as a result of dysregulation of channel expression and function without involving genetic alterations in calcium channel genes.3 For example, N- and T-type channels are upregulated in the primary afferent pain pathway during chronic pain states, and L-type channel dysregulation appears to be involved in Parkinson’s disease and drug addiction. In some cases, this can be rectified with pharmacological interventions. For example, the L-type channel blocker isradipine is in clinical trials for Parkinson’s disease and N-type channel blockers are approved for management of certain types of chronic pain.3 This adds to the wide use of dihydropyridines (DHPs) for
ll electrical activity in the mammalian brain, heart, and muscle relies on gradients of sodium, potassium, calcium, and chloride ions. Among these, calcium ions are privileged, because they fulfill a second critically important cell signaling function that includes the activation of calcium-dependent enzymes, gene transcription, muscle contraction, and secretion of neurotransmitters and hormones. As outlined in recent comprehensive review articles,1−3 one of the key pathways for entry of calcium ions from the extracellular space into the cytoplasm are voltage-gated calcium channels. These membrane proteins open in response to membrane depolarizations, and allow the influx of calcium ions along their electrochemical gradient. There are multiple types of calcium channels, and they can be divided into high and low voltage activated channels based on their voltage sensitivities. This high voltage activated calcium channels family includes L-type (Cav1.1-Cav1.4), P/Qtype (Cav2.1), N-type (Cav2.2), and R-type (Cav2.3) channels, which can be distinguished from each other based on pharmacological and biophysical characteristics.1,2 They each share a common subunit composition of a pore forming Cavα1 subunit plus ancillary Cavβ, Cavα2δ and in some cases Cavγ subunits, with the Cavα1 subunit being composed of four homologous membrane domains with six membrane spanning helices termed S1−S6.1,2 The low voltage activated channels include three different types of T-type (Cav3.1−Cav3.3) calcium channels, and contain only the Cavα1 subunit. These various calcium channels fulfill specialized physiological functions and specific cellular and subcellular distributions. For example, the Cav1.1 channels are expressed exclusively in skeletal muscle where they are essential for muscle contraction, whereas Cav2.2 channels are exclusively found in neurons where they trigger the release of neurotransmitters.2 Calcium channel function is regulated by a wide range of second messenger pathways, and via association with accessory proteins such as calmodulin. This occurs in a channel isoformdependent manner, thus providing specific cell types with many means of fine-tuning calcium signaling. Early studies on calcium channels focused on their biophysical and pharmacological properties, physiological roles, and their regulation in native tissues and cells. The cloning and functional expression of different calcium channel subtypes in the early to late nineties opened the field to the molecular dissection of calcium channel © XXXX American Chemical Society
Received: November 1, 2017 Accepted: November 3, 2017
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DOI: 10.1021/acschemneuro.7b00415 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience hypertension and the clinical use of T-type channel blockers for absence epilepsy.2,3 In this context, it is important to note that a given calcium channel isoform can have distinct drug interaction sites for different types of pharmacophores, and in many cases there are allosteric interactions between drug binding to these various sites. On the other hand, while there are specific inhibitors for particular calcium channel isoforms (which is especially true for peptide toxins such as ωconotoxins and ω-agatoxins isolated from venomous mollusks and spiders, respectively), many small organic inhibitors of calcium channels lack selectivity due to fact that there is a relatively high degree of sequence homology among members of the calcium channel family, and this has been a challenge for drug development.2 Two exciting studies published in Nature will greatly facilitate our understanding of drug interactions with calcium channels, and thus the development of new calcium channel inhibitors.4,5 Following their earlier description of calcium channel structure that was published in Science in 2015, a study by Wu and colleagues based on cryo-electron microscopy reported the structure of the rabbit skeletal muscle Cav1.1 calcium channel at a resolution of 3.6 Å in a complex with ancillary Cavβαδ subunits (Figure 1A).4 First, this work yielded important information into the structure and folding of the Cavα1 subunit, especially with respect to the positioning of the voltage-sensing domain of the channels in the closed (and potentially inactivated) conformation of the channel. Moreover, the structure revealed that the C-terminal domain of the channel engages in molecular interactions with the intracellular linker connecting domains III and IV of the channel structure to form a globular domain. The Cavα1 subunit structure also reveals a selectivity filter of calcium ions, as well as a gate, formed by a bundle of the four S6 transmembrane helices, that is involved in opening and closing of the permeation pathway for calcium ions. The structure provides new insights about the interactions of the Cavα1 subunit with ancillary Cavβ, Cavα2δ, and Cavγ subunits. Among these, the interactions with the Cavα2δ subunit are of particular importance, as this this subunit is the pharmacological target of gabapentinoid drugs.3 It consists of four cache domains and a von Willebrand factor domain A (VWA) that contains a metal ion binding site, and docks to three different extracellular loops on the Cavα1 subunit. The structure also confirms previous evidence that the Cavδ portion of this subunit is anchored to the extracellular leaf of the plasma membrane via glycophosphatidyl moiety attached to a cysteine residue. Altogether, this new structural information reveals unparalleled insights into the inner workings of the skeletal muscle L-type calcium channels that will not only offer the opportunity for rational drug design, but also offer the opportunity for homology modeling of other types of calcium channels. So how do L-type calcium channels interact with drugs? This was addressed by Tang and colleagues in a particularly clever manner.5 The authors mutated the bacterial sodium channel NavAb to create a calcium permeable version termed CavAb, and solved its structure using X-ray crystallography. CavAb does not exist in nature, and unlike voltage gated calcium channels (which have a four-repeat membrane domain structure1) it is composed of four identical single domain subunits. Nonetheless, for all intents and purposes, CavAb behaves in many ways like a voltage gated calcium channel, and can be inhibited by classical L-type calcium channel blockers such as DHPs and phenylalkylamines (PAAs). The authors
Figure 1. (A) Side view of the cryo-EM structure of the skeletal muscle Cav1.1 calcium channel in complex with ancillary Cavβ, Cavα2δ, and Cavγ subunits. Only two of the four transmembrane domains of the Cavα1 subunit are shown (domain II and IV), along with their membrane spanning helices. Note that the Cavα2δ docks to the extracellular side of the channel via its VWA domain whereas the Cavβ1a subunit binds at the cytoplasmic side. (B) Side view of CavAb based on X-ray crystallography depicting the interaction site for Brverapamil in the inner vestibule of the pore. The green circles reflect calcium ions whose permeation is blocked by the drug molecule. (C) Top view of CavAb depicting its interactions with membrane lipids (red, yellow) and a permeating ion (green circle) inside the pore cavity. In the absence of drugs (left panel), lipids occupy binding sites exposed to the plasma membrane, and four lipids (red) occupy sites around the pore of the channel containing its permeating ion (green dot). The DHP nimodipine (right panel) displaces one of the membrane lipids and mediates an allosteric change in the shape of the pore region, thus causing the pore bound lipids to rearrange and preventing ion permeation. Reprinted by permission from Macmillan Publishers Ltd.: Nature 2016 (panels (A) and (B) of the figure are from two different Nature publications, ref 4 for panel (A), and ref 5 for panel (B) and (C)).
then solved the crystal structure of CavAb in the presence of these compounds to learn how they interact with the channel (Figure 1B, C). In the case of DHPs, the authors showed that the DHP interaction site lies within the membrane phase. Despite the 4-fold symmetry of the channel, only one DHP molecule can be bound at a given time, as a result of allosteric interactions between the drug binding site and the remainder of the channel (Figure 1C). Interestingly, in the absence of DHPs, the binding site is occupied by membrane lipids, suggesting that this drug interaction site has the normal function of interacting with the plasma membrane. In contrast, the PAA Br-verapamil was shown to interact within the inner cavity of the permeation pathway of the channel by interacting with S6 helices (Figure 1B). As with DHPs, the interaction site is normally occupied by lipids. Not only does this work validate conclusions from early structure function and photoaffinity labeling studies, it provides key insights into the interaction of L-type calcium channel blockers with CavAb that are likely transferable to mammalian voltage-gated calcium channels. This, in turn, will help facilitate the creation of better, and perhaps more selective calcium channel antagonists for conditions such as hypertension. It is also likely that the CavAb model system may yield new insights B
DOI: 10.1021/acschemneuro.7b00415 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience into the interactions of calcium channels with other classes of drugs. Altogether, despite many years of progress and insights into calcium channel biology, nothing beats being able to look at them through structural information. For pharmacologists, these studies open a new window toward understanding of drug−channel interactions and thus new frontiers in drug discovery.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The author declares no competing financial interest.
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REFERENCES
(1) Simms, B. A., and Zamponi, G. W. (2014) Neuronal VoltageGated Calcium Channels: Structure, Function, and Dysfunction. Neuron 82, 24−45. (2) Zamponi, G. W., Striessnig, J., Koschak, A., and Dolphin, A. C. (2015) The Physiology, Pathology, and Pharmacology of VoltageGated Calcium Channels and Their Future Therapeutic Potential. Pharmacol. Rev. 67, 821−870. (3) Zamponi, G. W. (2016) Targeting voltage-gated calcium channels in neurological and psychiatric diseases. Nat. Rev. Drug Discovery 15, 19−34. (4) Wu, J., Yan, Z., Li, Z., Qian, X., Lu, S., Dong, M., Zhou, Q., and Yan, N. (2016) Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 Å resolution. Nature 537, 191−196. (5) Tang, L., Gamal El-Din, T. M., Swanson, T. M., Pryde, D. C., Scheuer, T., Zheng, N., and Catterall, W. A. (2016) Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs. Nature 537, 117−121.
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DOI: 10.1021/acschemneuro.7b00415 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
