Opinion
Voltage-gated calcium channels 2+ function as Ca -activated signaling receptors Daphne Atlas Department of Biological Chemistry, The Alexander Silverman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel
Voltage-gated calcium channels (VGCCs) are transmembrane cell surface proteins responsible for multifunctional signals. In response to voltage, VGCCs trigger synaptic transmission, drive muscle contraction, and regulate gene expression. Voltage perturbations open VGCCs enabling Ca2+ binding to the low affinity Ca2+ binding site of the channel pore. Subsequent to permeation, Ca2+ targets selective proteins to activate diverse signaling pathways. It is becoming apparent that the Ca2+-bound channel triggers secretion in excitable cells and drives contraction in cardiomyocytes prior to Ca2+ permeation. Here, I highlight recent data implicating receptor-like function of the Ca2+-bound channel in converting external Ca2+ into an intracellular signal. The two sequential mechanistic perspectives of VGCC function are discussed in the context of the prevailing and longstanding current models of depolarization-evoked secretion and cardiac contraction.
the ryanodine channel (RyR1) independently of Ca2+ conductance through the channel [2,3]. Similar to skeletal muscle, membrane depolarization triggers cardiac contraction by EC coupling, and synchronous transmitter release by ES coupling. However, because both processes require Ca2+ in the extracellular medium they do not seem to fit the skeletal muscle model of direct signaling. It was therefore natural to suggest that Ca2+ influx through the channel followed by Ca2+ binding to intracellular proteins is responsible for cardiac contraction and transmitter release. During the past two decades, functional and physiological studies have revealed a versatile signaling role of the VGCC [4,5] that is independent of ion flux [6–10]. It has been shown that the VGCC is a dynamic Ca2+-binding cell membrane protein that upon Ca2+ binding activates intracellular proteins prior to Ca2+ influx. This signaling activity implies receptor-like activity of the channel, with Ca2+ serving as the binding ligand.
Introduction When first characterized, VGCCs were viewed mainly as membrane proteins responsible for Ca2+ conduction into the cell. The proposal of a signaling role for the channel in driving contraction and triggering synaptic transmission, where it could act as a membrane receptor with Ca2+ as the binding ligand, was virtually overlooked. The prevailing mechanisms of excitation–contraction (EC) coupling and excitation–secretion (ES) coupling hold that intracellular Ca2+ ([Ca2+]i) is the trigger that drives cardiac contraction and synchronous transmitter release. VGCCs contribute to these mechanisms solely through elevating the intracellular concentration of calcium ions. In 1973, Schneider and Chandler suggested that voltage-dependent charge movement at the skeletal muscle Ltype calcium channel (Cav1.1) could initiate EC coupling [1]. Cav1.1 was later confirmed to physically interact with
The Ca2+-bound channel operates as a signal transduction receptor The process of depolarization-evoked release involves sequential activation of two Ca2+ binding proteins. Initially, Ca2+ binds to the selectivity filter of the VGCC that is exposed during membrane depolarization. Subsequent to permeation through the channel, Ca2+ binds to the vesicular synaptotagmin (syt1) protein. The conventional model suggests that Ca2+ binding to syt1 C2 domains triggers secretion, endorsing syt1 as the Ca2+ sensor protein of secretion [11–14]. However, the excitosome model suggests that the Ca2+-bound channel triggers secretion prior to Ca2+ influx. In this model, the Ca2+-bound channel that acts from within the excitosome complex (discussed later) serves as the Ca2+ sensor protein of synaptic transmission [6,8,9].
Corresponding author: Atlas, D. (
[email protected]). Keywords: transmitter release; synaptotagmin; syntaxin; SNAREs; ryanodine receptor; cardiac channel; cardiac contraction; RyR2. 0968-0004/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2013.12.005
The VGCC is a Ca2+ binding protein The Ca2+ binding site called the EEEE locus is generated by four glutamate residues located at the p-loop region of each of the four segments of the pore-forming subunit of the channel. Although the 3D structure of the channel is not available, the site has been modeled to denote two different affinities that define open and closed states of the channel [15]. The open state of the Ca2+ binding motif represents a multi-Ca2+ ion-occupied pore of low affinity (Kd13 mM) to Trends in Biochemical Sciences, February 2014, Vol. 39, No. 2
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Opinion enable ion flow. In the closed state, the channel pore is occupied with a single Ca2+ ion exhibiting high affinity (13 mM) Ca2+ binding site of the channel pore. The change in conformation induced by voltage perturbation and Ca2+ binding at the EEEE is transmitted via an intramembrane signaling to Sx1A. For simplicity, synaptosomal-associated-protein 25 (SNAP-25) and synaptotagmin (syt1) are not shown. (B) The top view of a hypothetical cluster, which, based on the nonlinear allosteric relationship of reconstituted secretion, was modeled to comprise three Sx1A and three Ca2+ channel molecules. For simplicity, SNAP-25 and syt1, which are part of the excitosome complex, are not shown. The large conformational changes can provide the energy required for the merger of two bilayers that ultimately culminate in the opening of the fusion pore (red circle). (C) It is hypothesized that fast opening of a fusion pore, outlined by Sx1A and channel molecules, is enabled by the intramembrane crosstalk between the Ca2+bound channel and Sx1A. Much like the opening of a camera lens, the opening and closure of the channel switches an on/off signal, providing a tight and rapid built-in initiation–termination mechanism.
Do Ca2+-bound channels also drive cardiac contraction? The crosstalk between Cav1.1 and RyR1 and between Cav1.2 and RyR2 controls the contractile function of the skeletal muscle and the heart, respectively. In the t-tubular membrane of the skeletal muscle, EC coupling is driven via protein–protein interaction between Cav1.1 (a11.1 subunit) and RyR1, independent of Ca2+-entry [38,71–74]. By contrast, in the cardiac cell, EC coupling is dependent on extracellular Ca2+. The consensus therefore is that [Ca2+]i elevated via Cav1.2 is indispensable for engaging Ca2+ efflux from RyR2 and eliciting EC coupling. The ability of Ca2+ to serve as an intrapore ligand at the VGCC, and act prior to permeation into the cytoplasm, inspired the exploration of a similar relationship between excitation and contraction in cardiac cells [10]. Specifically, Ca2+ binding at the channel as a potential trigger of EC coupling in cardiac myocytes was studied utilizing the impermeable nifedipine-insensitive Cav1.2 mutant a11.2/L775P/T1066Y (see above). Neonatal rat cardiomyocytes were infected with lentivirus encoding the non-conducting channel mutant, and Ca2+ transients were monitored at the readout of responses to membrane depolarization [10]. Much like the induction of synchronous release [40], membrane depolarization induced contraction despite the complete block of Ca2+ entry. The
data suggest that EC coupling can be triggered prior to intracellular Ca2+ binding to RyR2. This result was further confirmed by substituting the EEEE locus of the Ca2+-impermeable channel with AAAA, which eliminates Ca2+ binding at the pore [15]. When introduced into neonate cardiomyocytes, the resulting mutant a11.2/ AAAA/L775P/T1066Y lost the ability to support cardiomyocyte contraction [10]. Hence, occupancy of the EEEE low affinity Ca2+ binding site appeared essential for driving contraction. The data support a mechanism by which a conformational change is communicated to RyR2 through protein–protein interactions, most likely through the a11.2 II–III–loop [75], reminiscent of the a1II–III loop interactions with Sx1A and SNAP-25 [76,77]. The model combines membrane depolarization and Ca2+ dependency and can provide a highly regulated signal transduction pathway governed by protein–protein interaction as opposed to cations targeting RyR2 (Figure 4, left). These studies performed on neonatal cells, which do not have T-tubules, need to be demonstrated in mature cardiomyocytes (as hypothetically depicted in Figure 4, right; Box 1). The model implies that the initiation–termination kinetics of EC coupling is synchronized by the opening–closing kinetics of Cav1.2. Accordingly, upon return of Cav1.2 to its resting state, RyR2 resumes an inactive conformation. 49
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Current model Sarcolemma
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SR Cav1.2
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Figure 4. Hypothetical excitation–contraction (EC) coupling in cardiac cells. The current model (left). Initially, Ca2+ enters through Cav1.2 in the sarcolemma, followed by Ca2+ efflux from the sarcoplasmic reticulum (SR) via the ryanodine receptors (RyR) driving excitation–contraction (EC) coupling in cardiac cells (red rectangle). The release of Ca2+ from RyR, usually in clusters, activates the Ca2+induced Ca2+ release (CICR) process. This increase in cytosolic Ca2+ concentration triggers cardiac contraction. Termination of the signal according to this model is achieved with the decline in the intracellular Ca2+ within the SR. The deactivation of the RyR results in termination of Ca2+ release and prevention of contraction. The proposed model (right). Membrane depolarization opens the Cav1.2 pore and generates a transient active form of the channel. The Ca2+-bound active channel transmits a signal directly to the RyR, initiating Ca2+ release from the SR (red rectangle). Ca2+ efflux from RyR continues and the contractile proteins remain active as long as the Ca2+-bound channel is open. Upon returning to the resting state, Cav1.2 resumes an inactive closed state, and the disengagement of RyR2 and Cav1.2 stops RyR Ca2+ efflux. The model proposes that protein–protein interactions of Cav1.2 and RyR govern the activation and termination of heart contractile function. The proposed model is based on data derived from neonatal cells, which do not have T-tubules (rat cardiomyocytes develop T-tubules 2–3 weeks into the perinatal period); therefore, it remains to be shown for mature cardiomyocytes.
Terminating Ca2+ efflux from the RyR2 offers a putative mechanism of EC coupling signal termination. Based on electron microscopy studies, the cardiac Cav1.2 forms loose and irregular associations with the RyR2, as opposed to Cav1.1 association with RyR1 through the formation of tetrads [37]. It would be important to explore whether tetrad formation, which could enable a molecular coupling between Cav1.2 and RyR2 in mature cardimyocytes, requires Ca2+-bound Cav1.2. Interestingly, the formation of subcellular populations of Cav1.2, which each exhibit a unique regulatory role in cardiomyocytes [78], could result from variations in channel–RyR2 interaction, reminiscent of different pools of synaptic vesicles tethered to the membrane via the channel (see above). Concluding remarks When this field of investigation began, it was naturally presumed that Ca2+ entry through the VGCC resulting in elevated [Ca2+]i and binding to cytosolic proteins was indispensable for driving EC and ES coupling. What we have learned over the last two decades is that in addition to permeating Ca2+, the active form of the channel, namely the Ca2+-bound channel, can operate as a signaling membrane receptor to mediate ES and EC coupling prior to transporting Ca2+ into the cell. A transient Ca2+-bound channel that signals intracellular proteins from within an excitosome complex offers obvious spatial and temporal advantages over cytosolic Ca2+ targeting intracellular proteins. First, it enables signal transduction by conformational changes, providing for the fast kinetics of synaptic transmission (1 mM La3+ [81] and Fura-2 can detect 1–5 pM of La3+, this possibility is rather unlikely. What is the importance of Sx1A TMD in triggering transmitter release? The model of SNARE-mediated fusion indicates that proteins may operate by forcing lipid membranes close together without the need for a TMD-mediated perturbation [82]. These results conflict with mutations of the TMD of VAMP3 (the Sx1 in yeast vacuole fusion) that impair membrane fusion of yeast vacuoles [83] and impaired secretion observed with Sx1A TMD mutants [21,66–68]. Do Cav1.2 at T-tubules in mature cardiomyocytes organize with RyR2 and behave similar to Cav1.2 of neonatal cardiomyocytes? Even though Cav1.2 in the junctional domain opposite to the RyR2 lacks the tetrads found in skeletal muscle [84], cardiac EC coupling is controlled by Cav1.2 [85]. A direct coupling of Cav1.2 to RyR2 in mature cardiomyocytes and the possibility that Ca2+-bound Cav1.2 can induce tetrad formation needs to be demonstrated.
Opinion voltage relationship and the Ca2+ sensitivity of ES coupling. It provides the force needed for membrane merger and the opening of the fusion pore. The putative fusion pore outlined by Ca2+ channel and Sx1A molecules can hypothetically be rapidly opened similar to a camera lens. Fourth, it identifies the VGCC as the Ca2+ sensor of release and syt1 as the Ca2+ sensor of vesicle-priming that regulates the synaptic strength of neuronal cells by controlling the ratio of releasable/non-releasable pools. Finally, the direct interaction of the Ca2+-bound VGCC and RyR2 in cardiac cells can impart spatiotemporal setting for eliciting a high fidelity on/off switch of the contractile machinery by tightly controlling RyR2 Ca2+ efflux. A receptor-like function of the channel provides a novel insight into the mechanism of exocytosis and cardiac function. Further studies are required to fully understand the stoichiometry of Ca2+ channels and synaptic vesicles, and the precise molecular and functional features of these complex processes. Acknowledgments I graciously thank Thomas Schwarz, Marshall Devor, Robin Shaw, and Alex Levitzki for their suggestions and critical reading. I apologize to researchers whose work could not be cited owing to space limitations.
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