Calcium (Ca2+) entry via voltage-gated calcium channels (VGCCs), conveys the electric signals to intracellular transduction cascades in a wide variety of cells including neurons, muscle cells and endocrine cells1. Ca2+ dependent-signalling cascades are largely mediated by Ca2+ binding proteins2, 3, and are essential for multiple cellular and subcellular processes in physiological conditions. Perturbations of VGCCs functions can cause abnormity of cellular events, leading to pathological consequences. Ca2+ binding proteins mediate Ca2+-dependent signal transduction pathways and regulate Ca2+ influx via the VGCCs in Ca2+-dependent feedback mechanisms.

VGCCs are classified into L-, N-, P/Q-, R-, and T-types, based on their distinct electrophysiological and pharmacological properties4, 5. VGCCs are heteromultimeric protein complexes composed of a pore forming α1 and four distinct auxiliary subunits: α2, δ, β, and γ subunits4, 5, 6, 7. Mammalian α1 subunits are encoded by at least 10 distinct genes6, 7. The high voltage-activated VGCCs include CaV1 and CaV2 subfamilies. The CaV1 subfamily (CaV1.1 to CaV1.4) conducts L-type Ca2+ current and includes the channels containing α1S, α1C, α1D, and α1F subunits. The CaV2 subfamily (CaV2.1 to CaV2.3) conducts P/Q-type, N-type, and R-type Ca2+ currents, through the channels containing α1A, α1B, and α1E subunits, respectively. The CaV3 subfamily (CaV3.1 to CaV3.3) conducts low voltage-activated T-type Ca2+ current mediated by the channels containing α1G, α1H, and α1I subunits, respectively. The cell- and tissue-specific expression of these subunits allows for a vast variety of the channel subtypes exhibiting distinct functions.

Ca2+-binding proteins containing EF-hand Ca2+ binding motifs regulate mostly high voltage-activated VGCCs8, 9, 10, 11, 12. The EF-hand motif is a conserved Ca2+-binding structure, spanning a region of 30–35 amino acids containing a 12-residue Ca2+ binding loop flanked by the N- and C-terminal α-helix regions which are differentially exposed in the presence of Ca2+ 3, 13, 14. Each EF-hand protein has distinct Ca2+ binding affinity and cellular localization. The EF-hand Ca2+-binding protein superfamilies2, 3, 15, such as calmodulin (CaM), calcineurin, calcium binding proteins (CaBP), and neuronal Ca2+ sensors (NCSs), contains 2 to 4 functioning EF-hand Ca2+ binding domains. The EF-hand Ca2+-binding proteins may achieve their cellular effects through Ca2+-dependent or Ca2+-independent signalling mechanisms16, 17 (Figure 1). Many EF-hand Ca2+-binding proteins alter Ca2+ kinetics directly through regulation of VGCC properties8, 9, 10, 11, 12. With the availability of human genetic databases and advanced molecular technologies, growing evidences suggest that dysfunctions in Ca2+-binding protein mediated VGCC regulation may be one of the mechanisms leading to human diseases.

Figure 1
figure 1

Ca2+ binding proteins regulate voltage-gated Ca2+ channels (VGCCs) via Ca2+-dependent inactivation (CDI), Ca2+-dependent facilitation (CDF) and Ca2+-independent regulation (CIR) of the channels, hence contributing to Ca2+ homeostasis. Disrupting Ca2+-binding protein-mediated VGCC regulation results in pathophysiological processes leading to human diseases. CDI: Ca2+ ions entering the cell through VGCCs bind to Ca2+ binding proteins to (a) inactivate the channel via negative feedback mechanism, reducing further Ca2+ entry through the channel and (b) lead to downstream mechanisms and pathways implicated in human diseases. CDF: Ca2+ ions entering the cell through VGCCs bind to Ca2+ binding proteins to (c) facilitate the channel via a positive feedback mechanism, thus enhancing further Ca2+ entry through the channel and (d) lead to downstream mechanisms and pathways implicated in human diseases. CIR: Ca2+ binding proteins, in absence of Ca2+ binding (e) regulate VGCCs and (f) lead to downstream mechanisms and pathways implicated in human diseases.

Calmodulin mediated P/Q-type regulation in familial hemiplegic migraine type 1

The best studied Ca2+ binding protein that regulates VGCCs is CaM18, 19, 20. CaM contains 4 functional EF-hand motifs21, 22, and regulates VGCCs properties in an enzyme-inhibitor like fashion23. CaM binds to various high-voltage activated VGCCs and causes the Ca2+-dependent inactivation (CDI)8, 9, 24, 25 or Ca2+-dependent facilitation (CDF)10, 12, 26 (Figure 1). In brief, CaM has a higher binding affinity to Ca2+ in the N-lobe than the C-lobe EF-hand motifs. This allows for antagonistic regulation of the Ca2+ channel through differential Ca2+ binding to CaM27. Specifically, CDI of CaV1.2 channels8, 9, 24 and CDF of CaV2.1 channels depend on Ca2+ binding to the C-lobe of CaM10, 27. Conversely, Ca2+ binding to the N-lobe of CaM induces CDI of CaV2.110, 12, 28, CaV2.210, 12, and Cav2.310 type channels. The differential regulatory effects of CaM on VGCCs are likely due to different conformational changes in the structure of CaM following Ca2+ binding at alternate sites. CaM-mediated regulation of the presynaptic VGCCs results in a dual feedback regulation. The cellular and molecular mechanisms underlying CaM mediated VGCC regulation have been extensively reviewed previously18, 19, 20.

FHM is characterized by recurrent migraines and includes visual disturbance, sensory loss, hemiparesis and ataxia. FHM type 1 is an autosomal dominant type of migraine with aura and hemiparesis, which is linked to the VGCC α1-subunit gene, CACNL1A4 encoding CaV2.129, 30, 31. All five FHM1 mutations change the biophysical properties of CaV2.1 channels, leading to both gain and loss of P/Q-type channel function32, 33. Specifically, single channel recording showed that the mutations enhanced the open probability of the CaV2.1 channels and shifted the activation gating of the channel to more negative voltages, allowing increased Ca2+ influx at more negative membrane potentials in cerebellar neurons33, 34. Common treatments with Ca2+ channel blockers, such as verapamil, is effective in some FHM1 patients, carrying the CACNA1A mutations due to decreased open probability of P/Q-type CaV2.1 channels and reduced Ca2+ influx35.

Consistent with reports of increased open-channel probability32, 33, a recent study showed that FHM-1 missense mutants of the C-terminus in CaV2.1 subunit, R192Q and S218L, permitted a larger Ca2+ influx during action potentials than the wildtype channels in the cerebellar neurons36. Interestingly, these FHM-1 gain-of-function missense mutations characteristically occlude CDF of human CaV2.1 channels in both recombinant preparations and the cerebellar Purkinje cells. The altered CDF of CaV2.1 channels coincided with a decrease in short-term synaptic facilitation at the parallel fiber-to-purkinje cell synapse in the cerebellum in FHM-1 mutant mice36. The compelling evidence suggests that FHM-1 gain-of-function missense mutations of CaV2.1 channels favour a constitutively facilitated state that prevents further Ca2+-dependent CaM-mediated channel facilitation. It is hypothesized that disruption of CaV2.1 CDF may cause the cerebellar ataxia-associated FHM-1 due to an imbalance between excitatory and inhibitory inputs to the cerebellar Purkinje cells. This disruption suppresses the intrinsic pacemaker activity of these cells, thus leading to motor deficits36. The knock-in mouse model carrying FHM-1 R192Q mutation exhibited an enhanced velocity of cortical spreading depression in vivo34, and it is thus important to demonstrate whether the cortical hyper-excitability is also associated with perturbation of CDF of the mutant CaV2.1 in future studies.

CaBPs mediated L-type channel inactivation

CaBPs consist of 8 members (CaBP 1–8) and are considered similar to CaM in that they bear four recognizable, but not necessarily functional EF-hands37. CaBP1, also known as caldendrin (a splice variant of CaBP1)38, has 50% sequence homology to CaM and is widely expressed in the brain, including the cerebral cortex, hippocampus, in the cone bipolar and amacrine cells of the retina39, and in the inner hair cells. CaBP1 interacts with CaV2.1 P/Q -type channels40, 41, and L-type channels42. CaBP1 accelerates inactivation kinetics, prevents CaM-induced CaV2.1 channel facilitation, and shifts the voltage-dependent activation of CaV2.1 channels40. These effects of CaBP1 are mediated by binding to the CaM-binding IQ-domain in the α1A subunit of CaV2.1 channels. CaBP1 binding to the CaM binding domain (CBD) of α1A causes a significantly faster inactivation of CaV2.1 channel than CaM.

CaBPs regulate L-type channels in a Ca2+-independent manner40, 42, 43, 44 (Figure 1), in contrast to CaM. CaBP1 and CaBP4 act as negative regulators to compete with CaM binding to the C-terminal IQ motif in the CaV1.2 and CaV1.3 subunit42, 44, 45, 46. CaBP1 also interacts with the N-terminal domain of CaV1.2 to prolong the channel activation, independent of CaM effect42, 44. Some CaBPs, such as CaBP1 and CaBP4, have the capacity to negatively regulate influx of Ca2+ through a direct inhibitory interaction with plasma member P/Q-type channels in cochlear cells45, 46, 47. In the inner ear, at least 4 CaBPs have been found in hair cells, including CaBP1, CaBP2, CaBP4 and CaBP5. Sustained activation of presynaptic CaV1.3 channels triggers graded changes in neurotransmitter release which is required for sound detection46. CaBP1 binding to CaV1.3 channels on CaM interaction sites, induced a stronger, than CaBP4, inhibition of Ca2+-dependent channel inactivation46. Closely co-localization between CaBP1 and CaV1.3 at the presynaptic ribbon synapse of adult inner hair cells further suggests CaBP1-mediated inhibitory effect on Ca2+-dependent inactivation of CaV1.3 channel is critical for auditory transmission46.

CaBP448 and CaBP549 regulates L-type channels in photoreceptors. CaBP4 is located at the photoreceptor synaptic terminals in the retina, and is important for developing and sustaining synaptic transmission to bipolar cells43. CaBP4 regulates CaV1.4 channel and shifts the activation of CaV1.4 to more hyperpolarized potentials through a direct interaction with the C-terminal domain of the CaV1.4 channel protein. CaBP4−/− mice exhibited visual deficits similar to that caused by dysfunction of CaV1.4 channels43, 50, 51. CaBP4, like CaBP1, is found to interact with CaM-binding IQ domain in CaV1.3 to dampen the inactivation of the channel40, 46. CaBP4 has the capacity to eliminate even the baseline Ca2+ dependent inactivation of CaV1.345. Phosphorylation of S37 of CaBP4 by protein kinase Cζ in retina regulates CaV1.3, likely by facilitating the low-affinity interaction which exerts inhibitory regulation of CaV1.3 channel inactivation48. Phosphorylation of CaBP4 is critical for tuning presynaptic Ca2+ signals required for light-induced neurotransmitter release. Incomplete congenital stationary night blindness (CSNB2) is linked to mutations in both CaBP452, 53 and CaV1.454, 55, 56. Interrelation between CaBP4 and CaV1.4 in CSNB2 remains to be determined.

Bestrophin-1 mediated CaV1.3 modulation in macular degeneration

Bestrophins are a family of calcium-activated chloride channels57 encoded with VMD2 (Best vitelliform macular dystrophy-2) gene on chromosome 11q1358. Human bestrophin-1 (hBest1) is a founding member of the family and contains one EF-hand (EF1, 350–390) at the C-terminal and a regulatory domain adjacent to EF1 that is required for Ca2+ activation of the channel59. EF1 has a slightly higher Ca2+-binding affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. Mutations in hBest1 are involved in 100 human diseases58.

Retinal cell death, induced by glaucoma, diabetic reinopathy and age-related macular degeneration are primarily caused by a form of metabolic stress which results from a lack of nutrient supply. This process is initiated primarily through the activation of NMDA receptors with a subsequent influx of Ca2+ and Na+ ions into the cells60. The close relationship between ataxia and macular degeneration suggests that these disorders may share a common molecular network61. Oxidative stress, an important cause of retinal pigmental eipithelium death and subsequent age-related macular degeneration, induces calcium overload and leads to cell injury62. Oxidative stress induced elevation of Ca2+ level is sensitive to VGCC blocker62, suggesting the role of VGCCs in retinal cell death.

The hBest1 is localized at the basolateral plasma membrane of the retinal pigment epithelium cells63. Mutations of the hBest1 gene are associated with macular degeneration58. Bestrophin-1 is co-localized with CaV1.3 channels and the auxiliary β4-subunit in the cell membrane in the retinal pigment epithelium, and inhibits CaV1.3 channels via a direct interaction with the CaVβ4 subunit64, 65. Mutations of hBest1 on P330 and P334 prevented Best1-mediated inhibition of CaV1.364, 65. These findings provide new insights into the mechanisms of the retinal degeneration involved in hBest1-mediated CaV1.3 channel regulation.

Calcineurin regulation of Ca2+ channels in human diseases

Calcineurin is a calcium-dependent phosphatase activated by Ca2+/CaM66. It is a heterodimer and consisted of a 59 kDa catalytic subunit and a 19 kDa Ca2+-binding regulatory subunit. Calcineurin regulatory subunit is encoded with four putative EF-hand Ca2+-binding motifs33. The high-affinity Ca2+ binding site has a Kd of 24 nmol/L to Ca2+ whereas three low-affinity binding sites have a Kd of 15 μmol/L to Ca2+33. Calcineurin regulates L-type channels in both myocytes67 and neurons68, 69.

Calcineurin regulation of CaV1.2 L-type channel in cardiac hypertrophy

Ca2+ signalling pathways play a critical role in the development of cardiac hypertrophy, one of the predisposing factors related to hypertension and development of heart failure. The downstream effector of calcineurin, NFAT signalling transduction pathway, plays a critical role in pathological cardiac hypertrophy response70, 71. L-type CaV1.2 channels play an important role in blood pressure and development of myogenic tone. In cardiac muscles, L-type currents through CaV1.2 channels stimulate the excitation-contraction coupling. The C-terminus of this channel serves an autoinhibitory role to mediate the fight-or-flight response. Inactivation of CaV1.2 was found to reduce mean arterial blood pressure in mice and there was a severe dampening of response to penylephrine and angiotensin II, due to a significant portion of penylephrine-induced resistance being dependent on calcium influx through the CaV1.2 channel72. The truncation in the distal C-terminus of the α1 subunit of CaV1.2 leads to 10–15 fold increase in channel activity in mammalian cell lines73. The increased force of contraction during the fight-or-flight response is thought to be mediated by regulation of CaV1.2 channels via activation of secondary systems which act to phosphorylate the channel74. Deletion of this C-terminus causes a reduction in Ca2+ currents, as a result of lower surface expression of the channel, and leads to development of cardiac hypertrophy and premature death after E15 during embryonic development in mice25.

Recently, an EF-hand containing Ca2+ and integrin-binding protein-1 (CIB1) was found to specifically enhance cardiac pathological hypertrophy, without a role in altering physiological hypertrophy, through a regulation of calcineurin interaction with the sarcolemma75. One mechanism of calcineurin function is thought to be via L-type channels, which mediates Ca2+ influx into cardiomyocytes. Transgenic mice expressing an activated form of calcineurin were found to exhibit an enhanced ICa density compared with the non-transgenic littermates and to have a faster kinetics of ICa inactivation67. Calcineurin can directly bind to both N- and C-termini (a.a. 1943–1971) of CaV1.2 channels, and dephosphorylate the channels, which in turn increase the channel conductance76. Magnesium ions (Mg2+) bind to the C-terminal EF-hand to inhibit CaV1.2 channels, thereby reducing Ca2+ influx to maintain the intracellular Ca2+ at low levels77. Supplement of Mg2+ during global ischemia resulted in myocardial protection and improved functional recovery78. These evidences suggest that calcineurin serves as a key modulator of Ca2+-dependent pathways via regulation of CaV1.2 activities and in turn mediates the pathological electrical remodelling in cardiac hypertrophy.

Calcineurin regulation of L-type channels in neurodegenerative diseases

Calcineurin selectively enhances L-type channel activity in hippocampal neurons68, 69. Application of FK506, an inhibitor of calcineurin, reduces high-voltage-activated Ca2+ current via L-type, but not P/Q- or N-type channels68. PKA and calcineurin bind to A-kinase anchoring protein 79/150 (AKAP79/150), which interact with endogenous and recombinant CaV1.2 channels in hippocampal neurons and HEK293 cells, respectively66. Disruption of AKAP79/150-calcineurin anchoring increases Ca2+ current amplitude66. In contrast to CaM, calcineurin does not affect Ca2+-dependent inactivation of the neuronal L- or N-type channels; this conclusion is based on the findings that FK506 has no effect on the time-course of Ca2+ current inactivation of L-type channel in rat pituitary tumor cell line (GH3) and N-type channels in chicken dorsal root ganglion neurons, while Ca2+-dependent inactivation of the channels is prevented by Ca2+ chelator EGTA79. Calcineurin promotes dephosphorylation of 3′, 5′-cyclic AMP response element binding protein (CREB)29. Overexpression of calcineurin prevents30 and inhibition of calcineurin enhances long-term memory formation31, 80. The activity of calcineurin increases in the hippocampus during aging, and L-type channel block reduces calcineurin activity81. Cleavage of calcineurin by Ca2+-sensitive protease calpain82 enhances its phosphatase activity, which coincides with an increase in the number of neurofibrillary tangles in human brains of patients with Alzheimer's disease83. Interestingly, amyloid-β protein also increases the activity of calcineurin, leading to dephosphorylation of the proapototic protein BAD (Bcl-2/Bcl-XL-antagonist) causing cell death84 and subsequent activation of apoptotic pathways in Alzheimer's disease85. Calcineurin activity is implicated in age-related Ca2+ dysregulation in neurodegenerative disorders69. However, the role of EF-hand motifs in calcineurin-enhanced L-type channel activation, and the causal relation between calcineurin and VGCC regulation in degenerative disorders remain to be further investigated.

Perspectives and future directions

Functional diversity within related Ca2+-binding proteins may enhance the specificity of Ca2+ signalling by VGCCs in different cellular contexts. These channels undergo feedback mechanisms by Ca2+-dependent facilitation or inactivation. Such feedback is largely mediated by Ca2+ binding proteins. Increasing evidences demonstrate that the diverse and integrative roles of the abundant calcium binding proteins in VGCC regulation and Ca2+ signalling may be attributed to human diseases. However, our understanding of the role of such regulation in human diseases is rather limited, due to the complexity of the intracellular protein networks in which integrative functions of Ca2+ binding proteins must alter continuously to fit to the dynamic changes of Ca2+ signalling.

Many Ca2+ binding proteins have been found to regulate VGCCs, however, little is known about how such regulations are related to the pathophysiological processes. For instance, neuronal Ca2+ sensor-1/frequenin-1 (NCS-1/frq1) containing three functional EF-hand Ca2+ binding motifs15, 86, 87, 88 exhibits a 10 fold higher affinity for Ca2+ than CaM89. NCS-1 is highly localized at the presynaptic terminal of the vertebrates90, 91, 92, 93, 94, 95 and invertebrates88, 96, 97, 98, and facilitates synaptic transmission. It increases the P/Q-type Ca2+ current in the Calyx of Held of the giant presynaptic terminal90, and regulates the presynaptic N-type channels in motoneurons99 and growth cone VGCCs in Lymnaea neurons100, 101. Another example is visinin-like protein-2 (VILIP-2), a highly homologous subfamily of NCS proteins and capable of undergoing Ca2+-myristoyl switch102, 103. VILIP-2 slows inactivation104 and enhances facilitation105 of the presynaptic P/Q-type Ca2+ channels, by a direct interaction with the CBD of the C-terminus of CaV2.1. However, whether and how NCS-1 or VILIP-2-mediated VGCC regulation contributes to human diseases remain unclear. Conversely, down-regulation of VILIP-1 has been reported in several types of human cancers106, 107, and in heart failure/cardiac hypertrophy108. However, whether VILIP-1 effect is associated with VGCC regulation is unknown. Thus, it is necessary to further investigate if there is interrelation between VGCC regulation by Ca2+ binding proteins and human diseases.

Dysregulation of Ca2+ homeostasis leads to pathophysiological processes related to human diseases. For instance, a disruption of basal and stimulus-dependent Ca2+ levels has been reported in brains of patients suffering from Alzheimer's disease109. The level of Ca2+-sensitive protease calpain-1 in the prefrontal cortex is 3-fold higher in the postmorten brains of individuals with Alzheimer's disease, than those with other neurodegenerative disorders, such as Huntington's or Parkinson's disease. Calpain-1 activates Ca2+-sensitive phosphatase calcineurin by cleaving lysine501 at the C-terminal83. The abnormally enhanced calpain and truncated calcineurin activities correlate with the level of secreted amyloid precursor protein and progression of Alzheimer's disease110, 111. Thus, disruption of Ca2+ homeostasis in neuropathology of Alzheimer's disease may be mediated by hyperactivity of calpain-1 and calcineurin. Similarly, α-synuclein, a key protein in the pathophysiology of Parkinson's disease112, 113, binds to calmodulin in a Ca2+-dependent manner114. α-Synuclein-calmodulin interaction accelerates fibrilization of synuclein, crucial for forming the core of Lewy bodies. α-Synuclein also colocalizes with other Ca2+-binding proteins, including calbindin and parvalbumin115, implicating the significance of Ca2+-dependent signalling in the development of Parkinson's disease. One implication of these findings is that a tight regulation of Ca2+ homeostasis by Ca2+/Ca2+-sensitive proteins serves as a compelling mechanism for pathophysiological processes in neurodegenerative and/or cardiovascular disorders. Understanding such mechanisms allows us to identify potential drug targets for delaying or prevention of the onset of the related human diseases. However, this line of research is still at its infancy, and deserves further attention. With current advancement in genetic and epigenetic sequencing techniques and increased availability of the gene and protein databases of human diseases, exploring the role of Ca2+ binding proteins in VGCC regulation and their involvement in human diseases are becoming feasible in future studies.