T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis

T-type channels are transient low-voltage-activated (LVA) Ca²⁺ channels that control Ca²⁺ entry in excitable cells during small depolarizations around resting potential. Studies in the past 20 years focused on the biophysical, physiological, and molecular characterization of T-type channels in most tissues. This led to a well-defined picture of the functional role of LVA channels in controlling low-threshold spikes, oscillatory cell activity, muscle contraction, hormone release, cell growth and differentiation. So far, little attention has been devoted to the role of T-type channels in transmitter release, which mainly involves channel types belonging to the high-voltage-activated (HVA) Ca²⁺ channel family. However, evidence is accumulating in favor of a unique participation of T-type channels in fast transmitter release. Clear data are now reported in reciprocal synapses of the retina and olfactory bulb, synaptic contacts between primary afferent and second order nociceptive neurons, rhythmic inhibitory interneurons of invertebrates and clonal cell lines transfected with recombinant α₁ channel subunits. T-type channels also regulate the large dense-core vesicle release of neuroendocrine cells where Ca²⁺ dependence, rate of vesicle release, and size of readily releasable pool appear comparable to those associated to HVA channels. This suggests that when sufficiently expressed and properly located near the release zones, T-type channels can trigger fast low-threshold secretion. In this study, we will review the main findings that assign a specific task to T-type channels in fast exocytosis, discussing their possible involvement in the control of the Ca²⁺-dependent processes regulating exocytosis like vesicle depletion and vesicle recycling.     

CaV3.2 calcium channels control NMDA receptor-mediated transmission: a new mechanism for absence epilepsy

Ca_V3.2 T-type calcium channels, encoded by CACNA1H, are expressed throughout the brain, yet their general function remains unclear. We discovered that Ca_V3.2 channels control NMDA-sensitive glutamatergic receptor (NMDA-R)-mediated transmission and subsequent NMDA-R-dependent plasticity of AMPA-R-mediated transmission at rat central synapses. Interestingly, functional Ca_V3.2 channels primarily incorporate into synapses, replace existing Ca_V3.2 channels, and can induce local calcium influx to control NMDA transmission strength in an activity-dependent manner. Moreover, human childhood absence epilepsy (CAE)-linked hCa_V3.2(C456S) mutant channels have a higher channel open probability, induce more calcium influx, and enhance glutamatergic transmission. Remarkably, cortical expression of hCa_V3.2(C456S) channels in rats induces 2- to 4-Hz spike and wave discharges and absence-like epilepsy characteristic of CAE patients, which can be suppressed by AMPA-R and NMDA-R antagonists but not T-type calcium channel antagonists. These results reveal an unexpected role of Ca_V3.2 channels in regulating NMDA-R-mediated transmission and a novel epileptogenic mechanism for human CAE.     

Surface expression and function of Cav3.2 T-type calcium channels are controlled by asparagine-linked glycosylation

Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in T-type channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Ca_v3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Ca_v3.2 channel revealed that N-linked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Ca_v3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Ca_v3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca²⁺ current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.     

Cav3 T-type channels: regulators for gating,membrane expression,and cation selectivity

Ca_v3 T-type channels are low-voltage-gated channels with rapid kinetics that are classified among the calcium-selective Ca_v1 and Ca_v2 type channels. Here, we outline the fundamental and unique regulators of T-type channels. An ubiquitous and proximally located “gating brake” works in concert with the voltage-sensor domain and S6 alpha-helical segment from domain II to set the canonical low-threshold and transient gating features of T-type channels. Gene splicing of optional exon 25c (and/or exon 26) in the short III–IV linker provides a developmental switch between modes of activity, such as activating in response to membrane depolarization, to channels requiring hyperpolarization input before being available to activate. Downstream of the gating brake in the I–II linker is a key region for regulating channel expression where alternative splicing patterns correlate with functional diversity of spike patterns, pacemaking rate (especially in the heart), stage of development, and animal size. A small but persistent window conductance depolarizes cells and boosts excitability at rest. T-type channels possess an ion selectivity that can resemble not only the calcium ion exclusive Ca_v1 and Ca_v2 channels but also the sodium ion selectivity of Na_v1 sodium channels too. Alternative splicing in the extracellular turret of domain II generates highly sodium-permeable channels, which contribute to low-threshold sodium spikes. Ca_v3 channels are more ubiquitous among multicellular animals and more widespread in tissues than the more brain centric Na_v1 sodium channels in invertebrates. Highly sodium-permeant Ca_v3 channels can functionally replace Na_v1 channels in species where they are lacking, such as in Caenorhabditis elegans.     

Calcium-dependent inhibition of T-type calcium channels by TRPV1 activation in rat sensory neurons

We studied the inhibitory effects of transient receptor potential vanilloid-1 (TRPV1) activation by capsaicin on low-voltage-activated (LVA, T-type) Ca²⁺ channel and high-voltage-activated (HVA; L, N, P/Q, R) currents in rat DRG sensory neurons, as a potential mechanism underlying capsaicin-induced analgesia. T-type and HVA currents were elicited in whole-cell clamped DRG neurons using ramp commands applied before and after 30-s exposures to 1 μM capsaicin. T-type currents were estimated at the first peak of the I–V characteristics and HVA at the second peak, occurring at more positive potentials. Small and medium-sized DRG neurons responded to capsaicin producing transient inward currents of variable amplitudes, mainly carried by Ca²⁺. In those cells responding to capsaicin with a large Ca²⁺ influx (59% of the total), a marked inhibition of both T-type and HVA Ca²⁺ currents was observed. The percentage of T-type and HVA channel inhibition was prevented by replacing Ca²⁺ with Ba²⁺ during capsaicin application or applying high doses of intracellular BAPTA (20 mM), suggesting that TRPV1-mediated inhibition of T-type and HVA channels is Ca²⁺-dependent and likely confined to membrane nano-microdomains. Our data are consistent with the idea that TRPV1-induced analgesia may derive from indirect inhibition of both T-type and HVA channels which, in turn, would reduce the threshold of nociceptive signals generation (T-type channel inhibition) and nociceptive synaptic transmission (HVA-channels inhibition).     

T-type channels in the sino-atrial and atrioventricular pacemaker mechanism

Cardiac automaticity is a fundamental physiological function in vertebrates. Heart rate is under the control of several neurotransmitters and hormones and is permanently adjusted by the autonomic nervous system to match the physiological demand of the organism. Several classes of ion channels and proteins involved in intracellular Ca²⁺ handling contribute to pacemaker activity. Voltage-dependent T-type Ca²⁺ channels are an integral part of the complex mechanism underlying pacemaking. T-type channels also contribute to impulse conduction in mice and humans. Strikingly, T-type channel isoforms are co-expressed in the cardiac conduction system with other ion channels that play a major role in pacemaking such as f- (HCN4) and L-type Ca_v1.3 channels. Pharmacologic inhibition of T-type channels reduces the spontaneous activity of isolated pacemaker myocytes of the sino-atrial node, the dominant heart rhythmogenic centre. Target inactivation of T-type Ca_v3.1 channels abolishes I _Ca,T in both sino-atrial and atrioventricular myocytes and reduces the daily heart rate of freely moving mice. Ca_v3.1 channels contribute also to automaticity of the atrioventricular node and to ventricular escape rhythms, thereby stressing the importance of these channels in automaticity of the whole cardiac conduction system. Accordingly, loss-of-function of Ca_v3.1 channels contributes to severe form of congenital bradycardia and atrioventricular block in paediatric patients.     

Proliferation of human lens epithelial cells (HLE-B3) is inhibited by blocking of voltage-gated calcium channels

Calcium, as an integral part of a large number of cellular regulatory pathways, is selective in the control of specific cell functions like the start of G1 phase in cell cycle. Cell proliferation has been suggested to depend on increasing intracellular calcium levels. A major regulatory pathway for intracellular calcium is the calcium influx into the cell via voltage-gated calcium channels. T-type and L-type calcium channels are substantially present in human lens epithelial cell (hLEC), and total calcium currents are inhibited by mibefradil. Here, the hypothesis was tested if calcium influx via Ca_v channels regulates proliferation in epithelial cells. Cell proliferation was determined by cell culture assays using the L- and T-type Ca_v channel blockers mibefradil and verapamil as modulators for calcium influx. Calcium influx was investigated using the Manganese quench technique. Western blot experiments were accomplished under standard conditions using antibodies against MAPK 3. Mibefradil as well as verapamil impaired cell proliferation, but in different concentration ranges. Furthermore, the activation of MAPK 3 was reduced by both antagonists. Calcium influx was also reduced in the presence of both blockers. We conclude that the transmembrane influx of Ca²⁺ through Ca_v channels contributes to the regulation of hLEC proliferation, identifying Ca_v channel blockers as potential therapeutic substances in ocular diseases.     

ZD 7288 inhibits T-type calcium current in rat hippocampal pyramidal cells

Many studies have used the channel blocker ZD 7288 to assess possible physiological and pathophysiological roles of hyperpolarization-activated cation currents (I_h). In view of the known interplay between I_h and other membrane conductances, the effects in Wistar rats of ZD 7288 on low-voltage-activated (LVA (− or T-type)) Ca²⁺ channels were examined in whole-cell patch-clamp recordings from CA1 pyramidal cells in the presence of TTX, TEA, 4-AP, CsCl, BaCl₂ and nifedipine. ZD 7288 reduced T-type calcium channel currents and this effect was concentration dependant. ZD 7288 blocked T-type currents when applied extracellularly, but not when included in the recording pipette. Furthermore, ZD 7288 altered the steady-state voltage-dependent inactivation of T-currents. These results indicate that the blocker ZD 7288 has effects on voltage sensitive channels additional to those reported for the I_h current.     

Channelopathies in Cav1.1, Cav1.3, and Cav1.4 voltage-gated L-type Ca2+ channels

Voltage-gated Ca²⁺ channels couple membrane depolarization to Ca²⁺-dependent intracellular signaling events. This is achieved by mediating Ca²⁺ ion influx or by direct conformational coupling to intracellular Ca²⁺ release channels. The family of Ca_v1 channels, also termed L-type Ca²⁺ channels (LTCCs), is uniquely sensitive to organic Ca²⁺ channel blockers and expressed in many electrically excitable tissues. In this review, we summarize the role of LTCCs for human diseases caused by genetic Ca²⁺ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within their pore-forming α1 subunits causing hypokalemic periodic paralysis and malignant hyperthermia sensitivity (Ca_v1.1 α1), incomplete congenital stationary night blindness (CSNB2; Ca_v1.4 α1), and Timothy syndrome (Ca_v1.2 α1; reviewed separately in this issue). Ca_v1.3 α1 mutations have not been reported yet in humans, but channel loss of function would likely affect sinoatrial node function and hearing. Studies in mice revealed that LTCCs indirectly also contribute to neurological symptoms in Ca²⁺ channelopathies affecting non-LTCCs, such as Ca_v2.1 α1 in tottering mice. Ca²⁺ channelopathies provide exciting disease-related molecular detail that led to important novel insight not only into disease pathophysiology but also to mechanisms of channel function.     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.     

The genetic basis of Brugada syndrome: A mutation update

Brugada syndrome (BrS) is a condition characterized by a distinct ST‐segment elevation in the right precordial leads of the electrocardiogram and, clinically, by an increased risk of cardiac arrhythmia and sudden death. The condition predominantly exhibits an autosomal dominant pattern of inheritance with an average prevalence of 5:10,000 worldwide. Currently, more than 100 mutations in seven genes have been associated with BrS. Loss‐of‐function mutations in SCN5A, which encodes the α‐subunit of the Na_v1.5 sodium ion channel conducting the depolarizing I_Na current, causes 15–20% of BrS cases. A few mutations have been described in GPD1L, which encodes glycerol‐3‐phosphate dehydrogenase‐1 like protein; CACNA1C, which encodes the α‐subunit of the Ca_v1.2 ion channel conducting the depolarizing I_L,Ca current; CACNB2, which encodes the stimulating β2‐subunit of the Ca_v1.2 ion channel; SCN1B and SCN3B, which, in the heart, encodes β‐subunits of the Na_v1.5 sodium ion channel, and KCNE3, which encodes the ancillary inhibitory β‐subunit of several potassium channels including the Kv4.3 ion channel conducting the repolarizing potassium I_to current. BrS exhibits variable expressivity, reduced penetrance, and “mixed phenotypes,” where families contain members with BrS as well as long QT syndrome, atrial fibrillation, short QT syndrome, conduction disease, or structural heart disease, have also been described. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

State-dependent signaling by Cav1.2 regulates hair follicle stem cell function

The signals regulating stem cell activation during tissue regeneration remain poorly understood. We investigated the baldness associated with mutations in the voltage-gated calcium channel (VGCC) Ca_v1.2 underlying Timothy syndrome (TS). While hair follicle stem cells express Ca_v1.2, they lack detectable voltage-dependent calcium currents. Ca_v1.2^TS acts in a dominant-negative manner to markedly delay anagen, while L-type channel blockers act through Ca_v1.2 to induce anagen and overcome the TS phenotype. Ca_v1.2 regulates production of the bulge-derived BMP inhibitor follistatin-like1 (Fstl1), derepressing stem cell quiescence. Our findings show how channels act in nonexcitable tissues to regulate stem cells and may lead to novel therapeutics for tissue regeneration.     

CaV2.1 channelopathies

Mutations in the CACNA1A gene that encodes the pore-forming α₁ subunit of human voltage-gated Ca_V2.1 (P/Q-type) Ca²⁺ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2, and spinocerebellar ataxia type 6 (SCA6). For each channelopathy, the review describes the disease phenotype as well as the functional consequences of the disease-causing mutations on recombinant human Ca_V2.1 channels and, in the case of FHM1 and SCA6, on neuronal Ca_V2.1 channels expressed at the endogenous physiological level in knockin mouse models. The effects of FHM1 mutations on cortical spreading depression, the phenomenon underlying migraine aura, and on cortical excitatory and inhibitory synaptic transmission in FHM1 knockin mice are also described, and their implications for the disease mechanism discussed. Moreover, the review describes different ataxic spontaneous cacna1a mouse mutants and the important insights into the cerebellar mechanisms underlying motor dysfunction caused by mutant Ca_V2.1 channels that were obtained from their functional characterization.     

A molecule‐based genetic association approach implicates a range of voltage‐gated calcium channels associated with schizophrenia

Traditional genome‐wide association studies (GWAS) have successfully detected genetic variants associated with schizophrenia. However, only a small fraction of heritability can be explained. Gene‐set/pathway‐based methods can overcome limitations arising from single nucleotide polymorphism (SNP)‐based analysis, but most of them place constraints on size which may exclude highly specific and functional sets, like macromolecules. Voltage‐gated calcium (Ca_v) channels, belonging to macromolecules, are composed of several subunits whose encoding genes are located far away or even on different chromosomes. We combined information about such molecules with GWAS data to investigate how functional channels associated with schizophrenia. We defined a biologically meaningful SNP‐set based on channel structure and performed an association study by using a validated method: SNP‐set (sequence) kernel association test. We identified eight subtypes of Ca_v channels significantly associated with schizophrenia from a subsample of published data (N = 56,605), including the L‐type channels (Ca_v1.1, Ca_v1.2, Ca_v1.3), P‐/Q‐type Ca_v2.1, N‐type Ca_v2.2, R‐type Ca_v2.3, T‐type Ca_v3.1, and Ca_v3.3. Only genes from Ca_v1.2 and Ca_v3.3 have been implicated by the largest GWAS (N = 82,315). Each subtype of Ca_v channels showed relatively high chip heritability, proportional to the size of its constituent gene regions. The results suggest that abnormalities of Ca_v channels may play an important role in the pathophysiology of schizophrenia and these channels may represent appropriate drug targets for therapeutics. Analyzing subunit‐encoding genes of a macromolecule in aggregate is a complementary way to identify more genetic variants of polygenic diseases. This study offers the potential of power for discovery the biological mechanisms of schizophrenia.     

Transepithelial calcium transport in prolactin-exposed intestine-like Caco-2 monolayer after combinatorial knockdown of TRPV5, TRPV6 and Ca<Subscript>v</Subscript>1.3

The milk-producing hormone prolactin (PRL) increases the transcellular intestinal calcium absorption by enhancing apical calcium uptake through voltage-dependent L-type calcium channel (Ca_v) 1.3. However, the redundancy of apical calcium channels raised the possibility that Ca_v1.3 may operate with other channels, especially transient receptor potential vanilloid family calcium channels (TRPV) 5 or 6, in an interdependent manner. Herein, TRPV5 knockdown (KD), TRPV5/TRPV6, TRPV5/Ca_v1.3, and TRPV6/Ca_v1.3 double KD, and TRPV5/TRPV6/Ca_v1.3 triple KD Caco-2 monolayers were generated by transfecting cells with small interfering RNAs (siRNA). siRNAs downregulated only the target mRNAs, and did not induce compensatory upregulation of the remaining channels. After exposure to 600 ng/mL PRL, the transcellular calcium transport was increased by ~2-fold in scrambled siRNA-treated, TRPV5 KD and TRPV5/TRPV6 KD monolayers, but not in TRPV5/Ca_v1.3, TRPV6/Ca_v1.3 and TRPV5/TRPV6/Ca_v1.3 KD monolayers. The results suggested that Ca_v1.3 was the sole apical channel responsible for the PRL-stimulated transcellular calcium transport in intestine-like Caco-2 monolayer.     

Models of calcium permeation through T-type channels

Ca²⁺ entry is indispensable part of intracellular Ca²⁺ signaling, which is vital for most of cellular functions. Low voltage-activated (LVA or T-type) calcium channels belong to the family of voltage-gated calcium channels (VGCCs) which provide Ca²⁺ entry in response to membrane depolarization. VGCCs are generally characterized by exceptional Ca²⁺ selectivity combined with high permeation rate, thought to be determined by the presence in their selectivity filter of a versatile Ca²⁺ binding site formed by four glutamate residues (EEEE motif). The subfamily of LVA channels includes three members, Cav3.1, Cav3.2 and Cav3.3. They all possess two aspartates instead of glutamates (i.e., EEDD motif) in their selectivity filter and are the least Ca²⁺-selective of all VGCCs. They also have the lowest conductance, weakly discriminate Ca²⁺, Sr²⁺ and Ba²⁺ and demonstrate channel-specific sensitivity to divalent metal blockers, such as Ni²⁺. The available data suggest that EEDD binding site of LVA channels is more rigid compared to EEEE one, and their selectivity permeation and block are determined by two supplementary low-affinity intrapore Ca²⁺ binding sites located above and below EEDD locus. In addition, LVA channels have extracellular metal binding site that allosterically regulates channel’s gating, permeation and block depending on trace metals concentration.     

Introduction into Cav2.1 of the homologous mutation of Cav1.2 causing the Timothy syndrome questions the role of V421 in the phenotypic definition of P-type Ca2+ channel

The Timothy syndrome is a multisystem disorder associated with the mutation of a Gly residue (G402 or G406) in the Ca_v1.2 Ca²⁺ channel. G406 is localized at the end of the IS6 segment and just before the intracellular I–II loop, which is important for the regulation of channel inactivation and the binding of the Ca_vβ subunit. This Gly residue is conserved in all Ca_v1 and Ca_v2 channels, and the G to R exchange produces a strong decrease of inactivation not only in Ca_v1.2 but also in Ca_v2.3. Here, we show that the mutation into Arg or Glu of the homologous Gly residue in Ca_v2.1 (G363) produces also a slowing of inactivation. However, the G-to-A exchange that decreases the inactivation rate in Ca_v1.2 and Ca_v2.3 increases inactivation in Ca_v2.1. Each mutation affects specifically the gating properties of Ca_v2.1 that remain nevertheless modulated by the co-expressed β subunit as with wild-type channel. The strong decrease of inactivation produced by the G363R or G363E mutations was reminiscent to that previously described for a specific splice variant of Ca_v2.1 that contains a single Val residue inserted in the I–II loop (V421). We unexpectedly found that the V421 insertion does not affect the inactivation rate of Ca_v2.1 and that the effects previously attributed to this insertion, including those on G-protein regulation, can be reproduced by the G363E mutation. Altogether, our results highlight the role of G363 in gating properties, inactivation kinetics, and G-protein regulation of Ca_v2.1 and the lack of effect of V421 insertion on inactivation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CACNA1A haploinsufficiency causes cognitive impairment,autism and epileptic encephalopathy with mild cerebellar symptoms

CACNA1A loss-of-function mutations classically present as episodic ataxia type 2 (EA2), with brief episodes of ataxia and nystagmus, or with progressive spinocerebellar ataxia (SCA6). A minority of patients carrying CACNA1A mutations develops epilepsy. Non-motor symptoms associated with these mutations are often overlooked. In this study, we report 16 affected individuals from four unrelated families presenting with a spectrum of cognitive impairment including intellectual deficiency, executive dysfunction, ADHD and/or autism, as well as childhood-onset epileptic encephalopathy with refractory absence epilepsy, febrile seizures, downbeat nystagmus and episodic ataxia. Sequencing revealed one CACNA1A gene deletion, two deleterious CACNA1A point mutations including one known stop-gain and one new frameshift variant and a new splice-site variant. This report illustrates the phenotypic heterogeneity of CACNA1A loss-of-function mutations and stresses the cognitive and epileptic manifestations caused by the loss of Ca_V2.1 channels function, presumably affecting cerebellar, cortical and limbic networks.     

Ca2+ signaling by T-type Ca2+ channels in neurons

Among the major families of voltage-gated Ca²⁺ channels, the low-voltage-activated channels formed by the Ca_v3 subunits, referred to as T-type Ca²⁺ channels, have recently gained increased interest in terms of the intracellular Ca²⁺ signals generated upon their activation. Here, we provide an overview of recent reports documenting that T-type Ca²⁺ channels act as an important Ca²⁺ source in a wide range of neuronal cell types. The work is focused on T-type Ca²⁺ channels in neurons, but refers to non-neuronal cells in cases where exemplary functions for Ca²⁺ entering through T-type Ca²⁺ channels have been described. Notably, Ca²⁺ influx through T-type Ca²⁺ channels is the predominant Ca²⁺ source in several neuronal cell types and carries out specific signaling roles. We also emphasize that Ca²⁺ signaling through T-type Ca²⁺ channels occurs often in select subcellular compartments, is mediated through strategically co-localized targets, and is exploited for unique physiological functions. Lucius Cueni and Marco Canepari contributed equally to this review.