Block of T-type calcium channels by protoxins I and II
© Bladen et al.; licensee BioMed Central Ltd. 2014
Received: 4 March 2014
Accepted: 5 May 2014
Published: 9 May 2014
Low-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Therefore, finding specific modulators and/or blockers of T-type channels has become an important field of drug discovery. One characteristic of T-type calcium channels is that they share several structural similarities with voltage-gated sodium channels (VGSCs). We therefore hypothesized that binding sites for certain sodium channel blocking peptide toxins may be present in T-type calcium channels.
The sodium channel blocker ProTx I tonically blocked native and transiently expressed T-type channels in the sub- to low micro molar range with at least a ten-fold selectivity for the T-type calcium channel hCav3.1 over hCav3.3, and more than one hundred fold selectivity over hCav3.2. Using chimeras of hCav3.1 and hCav3.3, we determined that the domain IV region of hCav3.1 is a major determinant of toxin affinity, with a minor contribution from domain II. Further analysis revealed several residues in a highly conserved region between T-type and sodium channels that may correspond to toxin binding sites. Mutagenesis of several of these residues on an individual basis, however, did not alter the blocking effects of the toxin. ProTx II on the other hand preferentially blocked hCav3.2 and significantly shifted the steady state inactivation of this channel.
ProTx I blocks hCav3.1 both selectively and with high affinity. Domain IV appears to play a major role in this selectivity with some contribution from domain II. Given the structural similarities between sodium and T-type calcium channels and the apparent conservation in toxin binding sites, these data could provide insights into the development and synthesis of novel T-type channel antagonists.
KeywordsCalcium channels ProTx I ProTx II T-type blockers Electrophysiology
Low-voltage-activated (LVA) or “T-type” calcium channels are encoded by one of three different types of Cav3 α1 subunits (Cav3.1, Cav3.2 and Cav3.3, also known as α1G, α1H, and α1I, respectively) whose membrane topology is similar to those of sodium channels . They are activated by small membrane depolarizations and display rapid activation and inactivation kinetics  and they are responsible for triggering low-threshold depolarizations that in turn lead to the initiation of action potentials. Consequently, they are thought to be important for regulating neuronal and cardiac pacemaker activity, and disruption of their normal activity can contribute to cardiac hypertrophy [3–5] and neuronal hyperexcitabilty disorders such as epilepsy and pain [6–9]. Similarly, mutations in Cav3.2 T-type calcium channels have been linked to absence seizures [6, 7] and up-regulation of Cav3.2 T-type channel activity in primary afferent fibers has been linked to the development of chronic pain [8, 9]. Indeed, depletion of Cav3.2 results in hyposensitivity to pain [10, 11].
Modulators and blockers of T-type calcium channels may be useful in elucidating the exact role of these channels in cell signaling pathways and may be exploited for therapeutic purposes. Identification of drugs and molecules that selectively interact with T-type calcium channels has, however, so far proven difficult, although recently novel small organic scaffolds for T-type channel inhibitors have been derived from blockers of other calcium channel subtypes, such as L-type channels [12–14].
Another class of molecules that are known to be effective blockers of voltage gated ion channels are polypeptide toxins. The effects of toxins on ion channels have been extensively documented [for review see Catterall et al.,  and one toxin isolated from scorpion venom (kurtoxin) is known to be a potent blocker of T-type calcium channels . This toxin however has been shown to also block high-voltage activated calcium channels . More recently, two peptide toxins (ProTx I and ProTx II), isolated from Tarantula venom, have been shown to be potent blockers of both sodium and calcium channels [18–20]. Given the structural similarities between these two classes of ion channels, we tested to what extent these two toxins inhibited T-type calcium channels and identified channel structural determinants of toxin block.
Our results reveal that ProTx II selectively blocks human Cav3.2 (hCav3.2) albeit with far less efficacy than previous reports suggested . ProTx I on the other hand potently and preferentially blocks human Cav3.1 (hCav3.1) in the sub-micro molar range. We therefore focused on ProTx I and used chimeras of hCav3.1 and hCav3.3, as well as sequence alignment between T-type and Nav channels and toxin interaction sites, to determine that Domain IV of hCav3.1 and, to a lesser extent Domain II, are key toxin interaction regions.
Results and discussion
ProTx II is a preferential blocker of hCav3.2
Summary of biophysical parameters of various T-type calcium channels in the absence or presence of A, 1 μM ProTx II and B, 1 μM ProTx I
Current block (%) ProTx II (1 μM)
V0.5act (mV) Con
V0.5act (mV) ProTx II (1 μM)
Vh (mV) Con
Vh (mV) ProTx II (1 μM)
Current block (%) ProTxl (1 μM)
V 0.5 act (mV) Con
V 0.5 act (mV) ProTxl (1 μM)
Vh (mV) Con
Vh (mV) ProTxl (1 μM)
ProTx I is both a potent and selective blocker of hCav3.1
The domain IV region of hCav3.1 is important for ProTx I block and function of hCav3.1
Summary of biophysical parameters of human Cav3.1, rat Cav3.1 and hCav3.1-hCav3.3 chimeras in the absence or presence of 1 μM ProTx I
Current block (%) ProTx I (1 μM)
V0.5act (mV) Con
V0.5act (mV) ProTx I (1 μM)
Vh (mV) Con
Vh (mV) ProTx I (1 μM)
Substitution of individual amino acid residues in the putative toxin blocking sites do not affect ProTx I block of hCav3.1
A, Summary of biophysical parameters of hCav3.1 and hCav3.1 domain IV mutants and B, Summary of biophysical parameters of hCav3.1 domain II mutants in the absence or presence of 1 μM ProTx I
DOM IV mutant
IC50 Tonic ProTx I (μM)
V0.5act (mV) Con
V0.5act (mV) ProTx I (1μM)
Vh (mV) ProTx I (1 μM)
DOM II mutant
Current block (%) ProTx l (1 μM)
V 0.5 act (mV) Con
V 0.5 act (mV) ProTx I (1 μM)
Vh (mV) ProTx l (1 μM)
Comparison with previous work
Previous studies have shown that the tarantula venom peptides ProTx I and ProTx II inhibit voltage-gated sodium channels by shifting their voltage dependence of activation to more positive potentials [18, 20]. Our results show that ProTx I preferentially blocked hCav3.1 at sub-micro molar concentrations, but we did not observe any shift in half activation potential. Contrary to previous findings , ProTx II appeared to preferentially block hCav3.2. This toxin block caused a significant negative shift in half inactivation voltage of hCav3.2, but in contrast with previous studies on sodium and calcium channels, no significant change in half activation potential [18, 30, 31]. The apparent differences between some of our results and those of previous studies, may be in part be due to the different expression systems, recording methods and clones used. In previous studies, HEK cells and Xenopus oocytes were used to express rat Cav3 channels and toxin effect and channel kinetics were measured on tail currents as an indicator of potency. We attempted to address this discrepancy by using ProTx I on a rat Cav3.1 clone available to us and although our results showed a small positive shift in the voltage-dependence of activation [Table 2], it did not reach significance. Further experiments will need to be conducted to determine the precise biophysical interactions of this toxin with T-type calcium channels, and how toxin actions are affected by different experimental conditions.
Our data show that ProTx I and ProTx II potently and preferentially block hCav3.1 and hCav3.2 respectively. These two toxins block and modify T-type calcium channels using mechanisms similar to their interaction with sodium channels [18, 20]. Their effect on the voltage dependence of inactivation is reminiscent of β-scorpion toxin interactions with sodium channels . Overall, our data suggest that both ProTx I and ProTx II may be useful towards exploring the gating mechanisms of T-type calcium channels. Finally, the apparent similarities in the toxin binding sites between Nav and Cav channels may provide an insight into the synthesis of more potent antagonists that act on either or both of these channel subtypes.
Materials and methods
Human Cav3.2 cDNA was kindly provided by Dr. Terrance Snutch (University of British Columbia, Vancouver, Canada). Human Cav3.3 was obtained from Dr. Arnaud Monteil (CNRS Montpellier, France), human Cav3.1 was described previously by our laboratory  and human Cav3.1 and Cav3.3 chimeras were also described previously .
Unless stated otherwise, chemicals were purchased from Sigma (St. Louis, MO). Both ProTx I and ProTx II were purchased from Alomone Labs (Jerusalem, Israel) and were dissolved in external recording solution at the stock concentration of 1 mM. All subsequent dilutions were also made in external recording solution.
tsA-201 cell culture and transfection
Human embryonic kidney tsA-201 cells were cultured and transfected using the calcium phosphate method as described previously . Briefly, 6 μg of T-type calcium channel Cav3.1, Cav3.2, and Cav3.3, α1 subunits were transfected together with 0.5 μg Enhanced green fluorescent protein (EGFP) DNA (Clontech) as a marker. Cells were re-suspended with 0.25% (w/v) trypsin-EDTA (Invitrogen) and plated on glass cover slips a minimum of 3 to 4 hours before patching and kept at 37°C and 5% CO2.
Isolation of neurons
Thalamic neurons were isolated as described previously . Briefly, thalami of adult mice were dissected out, cut into small pieces and then digested in papain (Worthington, LS003126) containing culture media. After digestion, the tissue was washed and triturated for neuron dissociation. Thalamic neurons were then seeded at low density onto coverslips pretreated with poly-d-lysine (Sigma, P7280). Dorsal Root Ganglia (DRG) neurons were isolated as described previously . Briefly, DRG from adult mice were removed and placed in Ca2+ and Mg2+-free Hank’s Balanced Salt Solution, containing (in mM): 140 NaCl, 5.3 KCl, 0.4 KH2PO4, 0.3 Na2HPO4, 6 D-glucose, 10 HEPES, and 2 mg/mL collagenase (Type I, Worthington, Lakewood, New Jersey), and 200 units of DNaseI (Worthington, Lakewood, New Jersey). Ganglia were then incubated for 45 min at 37°C and subsequently placed in media supplemented with 10% fetal bovine serum to stop digestion. Cells were then dispersed with fire polished Pasteur pipettes and plated on glass coverslips coated with 100 μg/mL poly-L-lysine.
Whole-cell voltage-clamp recordings on tsA- 201 cells were performed at room temperature 2 to 3 days after transfection. Whole-cell voltage-clamp recordings on Neuronal cells were performed at room temperature, the following day after isolation. The external recording solution for all calcium channel recordings contained (in mM): 114 CsCl, 20 BaCl2, 1 MgCl2, 10 HEPES, 10 Glucose, adjusted to pH 7.4 with CsOH. For voltage-clamp recordings on neuronal cells, 5 μM CdCl2 was also added to the external solution to inhibit high voltage activated calcium channels. For all recordings, the internal patch pipette solution contained [in mM]: 108 CsMeSO4, 2 MgCl2, 11 EGTA, 10 HEPES adjusted to pH 7.3 with CsOH. The internal solution was supplemented with 0.6 mM GTP and 2 mM ATP, which were added directly to the internal solution immediately before use. Liquid junction potentials for the above solutions were left uncorrected. Recordings were digitized at 5 kHz and low-pass filtered at 1 kHz.
Toxins were prepared daily in external solution and applied locally to cells with the use of a custom built gravity driven micro-perfusion system that exchanges solution in approximately one second . Currents were elicited from a holding potential of −110 mV and were measured by conventional whole-cell patch clamp using an Axopatch 200B amplifier in combination with Clampex 9.2 software (Molecular Devices, Sunnyvale, CA). After establishment of the whole cell configuration, cellular capacitance was minimized using the analog compensation available on the amplifier. Series resistance was <10 MΩ and was compensated >85% in all experiments. Data were filtered at 1 kHz (8-pole Bessel) and digitized at 10 kHz with a Digidata 1320 interface (Molecular Devices). In addition to collecting the raw data, an online leak-subtraction protocol was used in which four pulses of opposite polarity and one-quarter amplitude were applied immediately before the test protocol. For current–voltage relation studies, the membrane potential was held at −100 mV and cells were depolarized from −80 to 20 mV in 10 mV increments. For steady-state inactivation studies, the membrane potential was depolarized by test pulses to −20 mV after 3.6-s conditioning pre-pulses ranging from −110 to −20 mV. The current amplitude obtained from each test pulse was then normalized to that observed at a holding potential of −110 mV.
Data analysis and statistics
Data were analyzed using Clampfit 9.2 (Molecular Devices). Preparation of figures and curve fitting was carried out with Origin 7.5 software (Northampton, MA, USA). Current–voltage relationships were fitted with the modified Boltzmann equation: I = (Gmax*(Vm-Erev))/(1 + exp((V0.5act-Vm)/ka)), where Vm is the test potential, V0.5act is the half-activation potential, Erev is the reversal potential, Gmax is the maximum slope conductance, and ka reflects the slope of the activation curve. Data from concentration-dependence studies were fitted with the equation y = A2 + (A1-A2)/(1 + ([C]/IC50)P) where A1 is initial current amplitude and A2 is the current amplitude at saturating drug concentrations, [C] is the drug concentration and P is the Hill coefficient. Statistical significance was determined by paired or unpaired Student’s t-Tests and one-way or repeated measures ANOVA followed by Tukey’s multiple comparison tests. Significant values were set as indicated in the text and figure legends. All data are given as means +/− standard errors. Steady-state inactivation curves were fitted using the Boltzmann equation: I = 1/(1 + exp((Vm-V h)/k)), where V h is the half-inactivation potential and k is the slope factor.
All experiments performed in this manuscript comply with the laws of Canada.
- ProTx I and II:
Protoxin I and II
Voltage gated calcium channel
Human voltage activated calcium channel
Voltage activated sodium channel
Half activation potential
Dorsal root ganglion.
This work was supported by a grant from the Canadian Institutes of Health Research. CB holds a studentship award from AIHS. IAS holds a Mitacs Elevate fellowship. GWZ is a Canada Research Chair and an Alberta Innovates-Health Solutions (AIHS) scientist.
- Catterall WA, Perez-Reyes E, Snutch TP, Striessnig J: International union of pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev. 2005, 57 (4): 411-25. 10.1124/pr.57.4.5.PubMedView ArticleGoogle Scholar
- Perez-Reyes E: Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev. 2003, 83: 117-161.PubMedView ArticleGoogle Scholar
- Bender KJ, Uebele VN, Renger JJ, Trussell LO: Control of firing patterns through modulation of axon initial segment T-type calcium channels. J Physiol. 2012, 590: 109-118.PubMedPubMed CentralView ArticleGoogle Scholar
- Cain SM, Snutch TP: Contributions of T-type calcium channel isoforms to neuronal firing. Channels. 2010, 4: 475-482.PubMedPubMed CentralView ArticleGoogle Scholar
- Cribbs L: T-type calcium channel expression and function in the diseased heart. Channels. 2010, 4 (6): 447-52.PubMedView ArticleGoogle Scholar
- Heron SE, Khosravani H, Varela D, Bladen C, Williams TC, Newman MR, Scheffer IE, Berkovic SF, Mulley JC, Zamponi GW: Extended spectrum of idiopathic generalized epilepsies associated with cacna1h functional variants. Ann Neurol. 2007, 62: 560-568. 10.1002/ana.21169.PubMedView ArticleGoogle Scholar
- Khosravani H, Zamponi GW: Voltage-gated calcium channels and idiopathic generalized epilepsies. Physiol Rev. 2006, 86: 941-966. 10.1152/physrev.00002.2006.PubMedView ArticleGoogle Scholar
- Altier C, Zamponi GW: Targeting Ca2+ channels to treat pain: T-type versus N-type. Trends Pharmacol Sci. 2004, 25: 465-470. 10.1016/j.tips.2004.07.004.PubMedView ArticleGoogle Scholar
- Park J, Luo ZD: Calcium channel functions in pain processing. Channels. 2010, 4 (6): 510-7.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang W, Gu J, Li YQ, Tao YX: Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain?. Mol Pain. 2011, 7: 16-10.1186/1744-8069-7-16.PubMedPubMed CentralView ArticleGoogle Scholar
- Cregg R, Momin A, Rugiero F, Wood JN, Zhao J: Pain channelopathies. J Physiol. 2010, 588 (11): 1897-1904. 10.1113/jphysiol.2010.187807.PubMedPubMed CentralView ArticleGoogle Scholar
- Lory P, Chemin J: Towards the discovery of novel T-type calcium channel blockers. Expert Opin Ther Targets. 2007, 11 (5): 717-722. 10.1517/1472822.214.171.1247.PubMedView ArticleGoogle Scholar
- Bladen C, Gündüz MG, Simşek R, Safak C, Zamponi GW: Synthesis and evaluation of 1,4-dihydropyridine derivatives with calcium channel blocking activity. Pflugers Arch. 2013, In press. doi:10.1007/s00424-013-1376-zGoogle Scholar
- Kumar PP, Stotz SC, Paramashivappa R, Beedle AM, Zamponi GW, Srinivasa A: Synthesis and evaluation of a new class of nifedipine analogs with t-type calcium channel blocking activity. Mol Pharmacol. 2002, 61: 649-658. 10.1124/mol.61.3.649.PubMedView ArticleGoogle Scholar
- Catterall WA, Cestèle S, Yarov-Yarovoy V, Yu FH, Konoki K, Scheuer T: Voltage-gated ion channels and gating modifier toxins. Toxicon. 2007, 49 (2): 124-41. 10.1016/j.toxicon.2006.09.022.PubMedView ArticleGoogle Scholar
- Chuang RS, Jaffe H, Cribbs L, Perez-Reyes E, Swartz KJ: Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin. Nat Neurosci. 1998, 1: 668-674. 10.1038/3669.PubMedView ArticleGoogle Scholar
- Sidach SS, Mintz IM: Kurtoxin, a gating modifier of neuronal high- and low-threshold ca channels. J Neurosci. 2002, 22: 2023-2034.PubMedGoogle Scholar
- Middleton RE, Warren VA, Kraus RL, Hwang JC, Liu CJ, Dai G, Brochu RM, Kohler MG, Gao YD, Garsky VM, Bogusky MJ, Mehl JT, Cohen CJ, Smith MM: Two tarantula peptides inhibit activation of multiple sodium channels. Biochemistry. 2002, 41 (50): 14734-47. 10.1021/bi026546a.PubMedView ArticleGoogle Scholar
- Xiao Y, Blumenthal K, Jackson JO, Liang S, Cummins TR: The tarantula toxins ProTx-II and huwentoxin-IV differentially interact with human Nav1.7 voltage sensors to inhibit channel activation and inactivation. Mol Pharmacol. 2010, 78 (6): 1124-34. 10.1124/mol.110.066332.PubMedPubMed CentralView ArticleGoogle Scholar
- Priest BT, Blumenthal KM, Smith JJ, Warren VA, Smith MM: ProTx-I and ProTx-II: gating modifiers of voltage-gated sodium channels. Toxicon. 2007, 49 (2): 194-201. 10.1016/j.toxicon.2006.09.014.PubMedView ArticleGoogle Scholar
- Hildebrand ME, Smith PL, Bladen C, Eduljeea C, Xie JY, Chen L, Fee-Maki M, Doering CJ, Mezeyova J, Zhu Y, Belardetti F, Pajouhesh H, Parker D, Arneric SP, Parmar M, Porreca F, Tringham E, Zamponi GW, Snutch TP: A novel slow-inactivation-specificion channel modulator attenuates neuropathic pain. Pain. 2010, 152 (4): 833-843.View ArticleGoogle Scholar
- Gui J, Liu B, Cao G, Lipchik AM, Perez M, Dekan Z, Mobli M, Daly NL, Alewood PF, Parker LL, King G, Zhou Y, Jordt SE, Nitabach MN: A tarantula-venom peptide antagonizes the trpa1 nociceptor ion channel by binding to the s1-s4 gating domain. Curr Biol. 2014, S0960-9822 (14): 00014-1. [Epub ahead of print]Google Scholar
- Bosmans F, Rash L, Zhu SY, Diochot S, Lazdunski M, Escoubas P, Tytgat T: Four novel tarantula toxins as selective modulators of voltage-gated sodium channel subtypes. Mol Pharmacol. 2006, 69 (2): 419-429.PubMedView ArticleGoogle Scholar
- Ohkubo T, Yamazaki J, Kitamura K: Tarantula toxin ProTx-I differentiates between human T-type voltage-gated Ca2+ Channels Cav3.1 and Cav3.2. J Pharmacol Sci. 2010, 112: 452-458. 10.1254/jphs.09356FP.PubMedView ArticleGoogle Scholar
- Kim D, Park D, Choi S, Lee S, Sun M, Kim C, Shin HS: Thalamic control of visceral nociception mediated by t-type ca2+ channels. Science. 2003, 302: 117-9. 10.1126/science.1088886.PubMedView ArticleGoogle Scholar
- Hamid J, Peloquin JB, Monteil A, Zamponi GW: Determinants of the differential gating properties of Cav3.1 and Cav3.3 T-type channels: a role of domain IV?. Neuroscience. 2006, 143 (3): 717-28. 10.1016/j.neuroscience.2006.08.023. 22PubMedView ArticleGoogle Scholar
- Rogers JC, Qu Y, Tanada TN, Scheuer T, Catterall WA: Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3-S4 extracellular loop in domain IV of the Na + channel alpha subunit. J Biol Chem. 1996, 271: 15950-15962. 10.1074/jbc.271.27.15950.PubMedView ArticleGoogle Scholar
- Wang J, Yarov-Yarovoy V, Kahn R, Gordon D, Gurevitz M, Scheuer T, Catterall WA: Mapping the receptor site for α-scorpion toxins on a Na + channel voltage sensor. Proc Natl Acad Sci U S A. 2011, 108: 15426-15431. 10.1073/pnas.1112320108.PubMedPubMed CentralView ArticleGoogle Scholar
- Gurevitz M: Mapping of scorpion toxin receptor sites at voltage-gated sodium channels. Toxicon. 2012, 60 (4): 502-11. 10.1016/j.toxicon.2012.03.022.PubMedView ArticleGoogle Scholar
- Smith JJ, Cummins TR, Alphy S, Blumenthal KM: Molecular interactions of the gating modifier toxin ProTx-II with NaV 1.5: implied existence of a novel toxin binding site coupled to activation. J Biol Chem. 2007, 282 (17): 12687-97. 10.1074/jbc.M610462200.PubMedView ArticleGoogle Scholar
- Edgerton GB, Blumenthal KM, Hanck DA: Inhibition of the activation pathway of the T-type calcium channel Ca(V)3.1 by ProTx II. Toxicon. 2010, 56 (4): 624-36. 10.1016/j.toxicon.2010.06.009.PubMedPubMed CentralView ArticleGoogle Scholar
- Beedle AM, Hamid J, Zamponi GW: Inhibition of transiently expressed low- and high-voltage-activated calcium channels by trivalent metal cations. J Membr Biol. 2002, 187: 225-238. 10.1007/s00232-001-0166-2.PubMedView ArticleGoogle Scholar
- Altier C, Khosravani H, Evans RM, Hameed S, Peloquin JB, Vartian BA, Chen L, Beedle AM, Ferguson SS, Mezghrani A, Dubel SJ, Bourinet E, McRory JE, Zamponi GW: ORL1 receptor-mediated internalization of N-type calcium channels. Nat Neurosci. 2006, 9: 31-40. 10.1038/nn1605.PubMedView ArticleGoogle Scholar
- Weiss N, Hameed S, Fernández-Fernández JM, Fablet K, Karmazinova M, Poillot C, Proft J, Chen L, Bidaud I, Monteil A, Huc-Brandt S, Lacinova L, Lory P, Zamponi GW, De Waard M: A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis. J Biol Chem. 2012, 287 (4): 2810-8. 10.1074/jbc.M111.290882.PubMedPubMed CentralView ArticleGoogle Scholar
- Feng ZP, Doering CJ, Winkfein RJ, Beedle AM, Spafford JD, Zamponi GW: Determinants of inhibition of transiently expressed voltage-gated calcium channels by ω-conotoxins GVIA and MVIIA. J Biol Chem. 2003, 2003 (278): 20171-20178.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.