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A potential role for T-type calcium channels in homocysteinemia-induced peripheral neuropathy

Gaifullina, Aisylu S.a,b; Lazniewska, Joannaa; Gerasimova, Elena V.b; Burkhanova, Gulshat F.b; Rzhepetskyy, Yuriya; Tomin, Andriya; Rivas-Ramirez, Paulaa; Huang, Juntingc; Cmarko, Leosa; Zamponi, Gerald W.c; Sitdikova, Guzel F.b; Weiss, Norberta,*

doi: 10.1097/j.pain.0000000000001669
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Homocysteinemia is a metabolic condition characterized by abnormally high level of homocysteine in the blood and is considered to be a risk factor for peripheral neuropathy. However, the cellular mechanisms underlying toxic effects of homocysteine on the processing of peripheral nociception have not yet been investigated comprehensively. Here, using a rodent model of experimental homocysteinemia, we report the causal association between homocysteine and the development of mechanical allodynia. Homocysteinemia-induced mechanical allodynia was reversed on pharmacological inhibition of T-type calcium channels. In addition, our in vitro studies indicate that homocysteine enhances recombinant T-type calcium currents by promoting the recycling of v3.2">Cav3.2 channels back to the plasma membrane through a protein kinase C–dependent signaling pathway that requires the direct phosphorylation of v3.2">Cav3.2 at specific loci. Altogether, these results reveal an unrecognized signaling pathway that modulates the expression of T-type calcium channels, and may potentially contribute to the development of peripheral neuropathy associated with homocysteinemia.

Homocysteine modulates T-type calcium channel expression through a protein kinase C–dependent signaling pathway and possibly contributes to homocysteinemia-induced mechanical allodynia.

aInstitute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republic

bDepartment of Human and Animal Physiology, Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russia

cDepartment of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada

*Corresponding author. Address: Norbert Weiss Institute of Organic Chemistry and Biochemistry, Flemingovo nam. 2, 16610 Prague, Czech Republic. Tel.: +420 220 183 464. E-mail address: weiss@uochb.cas.cz (N. Weiss).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

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1. Introduction

Homocysteinemia is a metabolic systemic disorder caused by defects in sulphur-containing amino acid (methionine and cysteine) metabolism, eventually leading to abnormal levels of non–building-block intermediary amino acid homocysteine.6 The physiological blood levels of homocysteine in healthy populations range from 10 to 12 μM and are determined primarily by the dietary intake of methionine, folate, and vitamin B12.30,52 By contrast, mild homocysteinemia is defined by homocysteine plasma levels above the range of 10 to 15 μM up to 25 μM, whereas patients with severe homocysteinemia can reach 500 μM.63 Elevated plasma level of homocysteine is recognized as an independent risk factor for cardiovascular disorders.25 In addition, elevated homocysteine is observed in a number of human neurological conditions including peripheral neuropathy (PN).2 For instance, clinical studies have revealed that homocysteine increases the prevalence of peripheral diabetic neuropathy (PDN) and exacerbates any preexisting PDN.1,11,15 The prevalence for PDN is increased by about 7% for each 1 μmol/L increase in plasma homocysteine.41 It was also reported that homocysteine is a direct risk factor for PN.41 Furthermore, it was evidenced that homocysteinemia is associated with anti-Parkinson's disease medication, and an increased prevalence for PN is observed in patients on L-DOPA medication,45 further supporting a link between homocysteine and the development of PN. However, despite the growing body of evidence relating homocysteinemia with PN, there is a dearth of knowledge regarding the cellular and molecular roles of homocysteine in the development of PN.

It is well known that within neurons of the dorsal root ganglia (DRG) and spinal cord, voltage-gated calcium channels (VGCCs) play an essential role in the processing of peripheral nociception.10,69 Although DRG neurons express a variety of VGCCs, the high-voltage-activated (HVA) Cav2.2 (N-type) and low-voltage-activated (LVA) v3.2">Cav3.2 (T-type) channels are the main contributors to the processing of the pain signals. For instance, Cav2.2 channels expressed in presynaptic terminals of afferent nerve fibers support the release of pronociceptive neurotransmitters such as glutamate, substance P, and calcitonin gene-related peptide.22,42,60 By contrast, axonal v3.2">Cav3.2 channels contribute to the excitability of afferent fibers along with voltage-gated sodium channels.29 Expression of v3.2">Cav3.2 channels was also reported in nerve endings in skin hair follicles57 where they support the transmission of low-threshold mechanical signals24,28 and also contribute to the pathological development of mechanical allodynia.24,47 Finally, there is a body of evidence implicating v3.2">Cav3.2 channels in excitatory synaptic transmission in the dorsal horn of the spinal cord,26,32,64,72 possibly by virtue of their coupling with the vesicular release machinery of neurotransmitters.71 However, the role of VGCCs in the development of homocysteinemia-induced PN has never been explored. Here, using a rat model of prenatal homocysteinemia, we demonstrate that homocysteine is associated with the development of mechanical allodynia and can be reversed by pharmacological inhibition of T-type calcium channels. In addition, we show that recombinant v3.2">Cav3.2 channels are upregulated in the presence of homocysteine. This regulation relies on the direct phosphorylation of v3.2">Cav3.2 by protein kinase C (PKC), which in turn potentiates the recycling of the channel back to the cell surface leading to an increased density of v3.2">Cav3.2 in the plasma membrane.

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2. Methods

2.1. Experimental model of prenatal hyperhomocysteinemia

All experiments were performed in accordance with the European directive 2010/63/EU for animal experimentation and with the local ethical committee of the Kazan Federal University (protocol No 8 from May 5, 2015). Adult Wistar rats were kept on a 12-hour light/dark under controlled temperature (22-24°C) with ad libitum access to food and water. Experimental homocysteinemia was induced as previously described.18,27,77 Briefly, adult Wistar females were fed with either a regular-based diet (control), or supplemented daily with methionine (7.7 g/kg body weight) to induce homocysteinemia, starting 3 weeks before gestation up to 2 weeks after delivery. Behavioral experiments were performed on offspring at the age of 4 to 5 months.

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2.2. Measurement of plasma homocysteine level

Blood was drawn by cardiac puncture under isoflurane anesthesia as previously described.5 Blood samples were centrifuged at 3000 rpm for 15 minutes, and plasma homocysteine level was determined by electrochemical detection using a nanocarbon modified electrode as previously described.38,39,82

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2.3. Measurement of nociceptive behavior

Mechanical nociception was assessed using a series of calibrated Von Frey filaments with strength ranging from 0.008 to 0.6 g of target force, corresponding to 2.53 to 18.4 g/mm2 pressure. Animals were placed individually in an acrylic cage with a mesh floor and allowed to acclimatize for 1 hour until major grooming and exploration activities have ceased. The “ascending stimulus” method was used to estimate the mechanical withdrawal threshold as previously described.19 Filaments, tested in order of increasing stiffness, were applied 5 times, perpendicularly to the plantar surface of hind paw and pressed until they bent. The first filament that evoked at least one response was assigned as the threshold.65 After determination of individual thresholds, 50 μL of the T-type channel blockers TTA-A2 (2.6 μM), ML218 (1 nM), or Mibefradil (5 nM) was injected under the skin of the plantar surface of the right hind paw, whereas the left hind paw was injected with 50 μL of the corresponding vehicle solution, and mechanical nociception was reassessed 20 and 60 minutes after injection.

Thermal nociception was examined by measuring the latency to hind paw licking on a thermostatically heated surface maintained at the constant temperature of 52°C as previously described.21

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2.4. Dorsal root ganglia neuron culture

Dorsal root ganglia neurons were isolated enzymatically from 5- to 6-week-old mice as previously described.56 Cells were seeded in Poly-L-Lysine-coated dishes (Sigma-Aldrich P4707, St. Louis, MS) in DMEM medium (Thermo-Fisher 31966, Waltham, MA) supplemented with 10% FBS (Thermo-Fisher 10500064) and penicillin (100 units/mL)-streptomycin (100 μg/mL) (Thermo-Fisher 15140122).

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2.5. Heterologous expression

The human HA-tagged v3.2">Cav3.2 channel was expressed in human embryonic kidney tsA-201 cells as previously described.58 The CHO cell line stably expressing the rat Cav2.2-EGFP/α2δ-1/β channel complex was previously described.43 Cells were grown in standard conditions at 37°C in a controlled atmosphere containing 5% CO2.

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2.6. Patch clamp electrophysiology

Recording of calcium currents in DRG neurons was performed after 24 hours in culture in a bath solution containing (in millimolar): 5 CaCl2, 5 KCl, 1 MgCl2, 128 NaCl, 10 TEA-Cl, 10 D-glucose, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 0.0005 TTX (pH7.2 with NaOH). Patch pipettes (2-4 MΩ) were filled with a solution containing (in millimolar): 110 CsCl, 3 Mg-ATP, 0.5 Na-GTP, 2.5 MgCl2, 5 D-glucose, 10 EGTA, and 10 HEPES (pH7.4 with CsOH). For recording of sodium currents, the bath solution contained (in millimolar): 35 NaCl, 30 TEACl, 65 choline-Cl, 5 MgCl2, 10 HEPES, 10 D-glucose, and 0.05 CdCl2 (pH7.2). The pipette solution contained (in millimolar): 100 CsCl, 40 TEA-Cl, 5 NaCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 2 MgATP, and 1 Li2GTP (pH7.2). Patch clamp recording was performed on small-size DRG neurons (15-30 μm diameter) that were selected using an ocular micrometer.

Recording of calcium currents in tsA-201 cells was performed 72 hours after transfection. The voltage dependence of the peak current density was fitted with the modified Boltzmann Equation 1:where I(V) is the peak current amplitude at the command potential V, Gmax the maximum conductance, Vrev the reversal potential, V0.5 the half-activation potential, and k the slope factor. The voltage dependence of the calcium conductance was calculated using the modified Boltzmann Equation 2:where G(V) is the calcium conductance at the command potential V.

Whole-cell recordings were performed at room temperature (22-24°C) using an Axopatch 200B amplifier (Axon Instruments). Acquisition and analysis were performed using pClamp 10 and Clampfit 10 software, respectively (Axon Instruments).

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2.7. Nonstationary noise analysis

Nonstationary noise analysis of v3.2">Cav3.2 channels expressed in tsA-201 cells was performed from a set of 100 current traces recorded in response to 150-ms long depolarizing step to −20 mV from a holding potential of −100 mV as previously described.53 The variance-mean analysis was performed using ANA software developed by Dr. Michael Pusch at the Institute of Biophysics in Genova, Italy (http://users.ge.ibf.cnr.it/pusch/programs-mik.htm). The fit of the variance-mean histogram is performed with the following Equation 3:where σ02 is the background variance, leak is the leak current, i is the single channel current, N the number of functional channels, o is the “fraction open channel noise” (normally = 0), and I the independent variable, the mean current.54

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2.8. Plasmids cDNA constructs

The human wild-type HA-tagged v3.2">Cav3.2 construct (HA-v3.2">Cav3.2WT) was previously described.20 This plasmid was used as a template to generate phosphorylation-deficient channel mutants by PCR mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB) and the following pairs of primers: S532A: 5′-CTACCATTTCgcCCATGGCAGCCCCC-3′ (forward) and 5′-TGGTGGTGGTGGTGGTGA-3′ (reverse); S653A: 5′-GAGCTTGAACgcCCCTGATCCCTACGAGAAGATCCCGCATGTGG-3′ (forward) and 5′-AACGGGCCGTGCCCCCCG-3′ (reverse); S1144A: 5′-CCGGCGCTCCgcCTGGAGCAGC-3′ (forward) and 5′-CTGCTCCAGGCGCCACTG-3′ (reverse); S2188A: 5′-GAAGAAGATGgcCCCCCCCTGCATCTCG-3′ (forward) and 5′-TTTCTGCGCGCACCCAGA-3′ (reverse). The following pair of primers was used to generate the redox-insensitive v3.2">Cav3.2 H191Q mutant: 5′-GTTGGACGGACAaAACGTGAG-3′ (forward) and 5′-GAGTACTCCATCATGCCC-3′ (reverse). All final constructs were verified by sequencing of the full-length coding sequence. The mCherry-tagged v3.2">Cav3.2 construct along with the ER- and Golgi-targeted EGFP constructs were previously described.36

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2.9. SDS-PAGE and immunoblot analysis

Total lysates were prepared from tsA-201 cells expressing HA-tagged v3.2">Cav3.2 channels. Cells were lysed in NP40 lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40, pH 8.0), and the protein concentration was estimated using a Bradford Protein Assay (BioRad). Total lysate (25 μg) were separated on 5% to 20% gradient SDS-PAGE in Laemmli buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) for 2 hours at 30 mA and transferred for 2 hours at 100 V onto 0.45-μm Hybond PVDF membrane (Amersham 10600023) in a transfer buffer containing (in mM): 25 Tris, 192 glycine, and 20% methanol, pH 8.3. For detection of the HA-hCav3.2 channel, the membrane was incubated with a primary rat monoclonal anti-HA antibody (clone 3F10 Roche 119867423001) diluted at 1:1000 and incubated with a secondary HRP-conjugated antibody (Jackson ImmunoResearch 112-035-003, West Grove, PA) diluted at 1:10,000. The membrane was treated with enhanced chemiluminescence buffer (0.1 M Tris-HCl pH 8.5 containing 0.2 mM p-coumaric acid [Roth 9908.1], 1.25 mM luminol [Roth 4203.2], and 0.01% hydrogen peroxide [Roth 8070.2]). Immunoreactive bands were detected with ImageQuant LS 4000 (Amersham 28955810).

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2.10. Surface immunostaining

Immunostaining of HA-tagged v3.2">Cav3.2 channels in tsA-201 cells was performed using a primary monoclonal mouse anti-HA antibody (Abcam ab18181, Cambridge, United Kingdom) and a secondary goat polyclonal anti-mouse Alexa488-conjugated antibody (Jackson ImmunoResearch 115-545-003) as previously described.36 Surface expression of the channel was assessed from low-magnification confocal images acquired with a Zeiss LSM780 microscope and an oil immersion Zeiss × 63/1.4 plan apochromatic objective and analyzed with ImageJ software.

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2.11. Measurement of protein kinase C activity

Protein kinase C activity in tsA-201 cells exposed to elevated levels of Hcy was evaluated using PepTag nonradioactive assay (Promega V5330, Madison, WI) according to the manufacturer's instructions. Cells were washed twice with PBS and dissociated with Versene solution (Gibco, 15040066, Waltham, MA), and resuspended in the extraction buffer (25 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mM β-mercaptoethanol, 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 0.5 mM PMSF) and lysed with precooled Dounce homogenizer on ice. Lysates were centrifuged for at 4°C for 5 minutes at 14,000g, and the supernatant was applied to DEAE-cellulose column, preequilibrated with the same extraction buffer. For elution of PKC-enriched fraction, a 200-mM NaCl solution was added to the extraction buffer. All purification steps were performed at 4°C. The protein concentration for each effluent was measured by Bradford protein assay, and the equivalent of 10 µg of protein per sample was used for further analysis. The reaction mixture (25 µL) containing 1 µg of PepTag C1 peptide (PLSRTLSVAAK) in PepTag PKC Reaction Buffer (20 mM HEPES, 1.3 mM CaCl2, 1 mM DTT, 10 mM MgCl2, and 1 mM ATP, pH 7.4) was preheated for 2 minutes at 30°C, and mixed with the protein extract samples. After 30-minute incubation at 30°C, the reaction was terminated by heating to 95°C for 10 minutes. After addition of 1 µL of glycerol, samples were loaded on a 0.8% agarose gel and separated by horizontal electrophoresis in a running buffer containing 50 mM Tris-HCl, pH 8.0 for 30 minutes at 100 V. Autoluminescence of the PepTag C1 peptide was detected using a transilluminator and quantified using ImageJ software.

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2.12. Chemicals

L-homocysteine (Sigma 69453) was dissolved in H2O to prepare a stock solution of 300 mM, and was diluted in the cell culture medium at a final concentration of 300 μM for 24 hours unless stated otherwise. L-glutamic acid (L-Glu, Sigma G1251) was dissolved at 10 mM in 1M HCL and used at a final concentration of 10 μM. 2-methylaminoisobutyric acid (MEA) was prepared at 20 mM in H2O and used at 10 μM final. N-acetylglucosamine (NAC) was prepared freshly at 10 mM in H2O and used at 1 mM final. Phorbol 12-myristate 13-acetate (PMA, Sigma P8139) was dissolved in DMSO and used at 10 nM final. Mibefradil (Sigma M5441) was dissolved in sterile saline solution (0.9% NaCl) and used at a final concentration of 5 nM. ML218 (Sigma SML0385) was dissolved in DMSO and used at a final concentration of 1 nM. TTA-A2 (Alomone Labs T-140) was dissolved in DMSO and used at a final concentration of 2.6 μM.

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2.13. Statistical analysis

Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc). Comparison between 2 groups was performed using a Mann–Whitney test unless stated otherwise in the article. Data are presented as mean ± SEM for n experiments. Data were considered statistically significant for P < 0.05, and the following significance values are given: *P < 0.05, **P < 0.01, and ***P < 0.001. NS, not significant.

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3. Results

3.1. Homocysteinemia induces mechanical allodynia

To establish whether homocysteinemia modulates pain signaling, we assessed nociceptive behavior in a rat model of prenatal homocysteinemia (Fig. 1A). Maternal homocysteinemia was induced with methionine-enriched diet resulting in a ∼3-fold increase in plasma homocysteine concentration (21.5 ± 3.1 µM, n = 4) compared to control females (7.1 ± 0.6 µM, n = 4) (Fig. 1B). Behavioral experiments were performed on offspring at the age of 4 to 5 months where plasma homocysteine concentration was increased by ∼2.4-fold (P = 0.0159) in offspring with maternal homocysteinemia (18.6 ± 3.2 µM, n = 4) compared to offspring from control females (7.9 ± 1.7 µM, n = 5), comparable to mild homocysteinemia in clinic (Fig. 1B). To determine whether pain signaling is altered during homocysteinemia, we assessed both mechanical nociception and thermal nociception. Using a large cohort of animals, we observed that mechanical withdrawal threshold was decreased by 1.8-fold (P < 0.0001) in animals with homocysteinemia (5.3 ± 0.1 g/mm2, n = 96) compared to control animals (9.4 ± 0.6 g/mm2, n = 96) (Fig. 1C). By contrast, we did not observe significant alterations (P = 0.1713) of thermal nociception (Fig. 1D).

Figure 1.

Figure 1.

Taken together, these data indicate that peripheral nociception is altered during homocysteinemia and support a causal link between homocysteinemia and the development of mechanical allodynia.

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3.2. Homocysteine enhances T-type currents in dorsal root ganglia neurons

It is well recognized that several VGCCs encode peripheral nociceptive signaling in the DRG. To test whether homocysteine modulates native VGCCs, we performed whole-cell patch clamp recordings on cultured adult small DRG neurons (15-30 μm diameter corresponding to unmyelinated nociceptive C fibers76) exposed for 24 hours to homocysteine. Consistent with previous studies,9 we observed different electrophysiological profiles of small DRG neurons with regards to their voltage-activated calcium conductances. A subset of cells displayed a combination of (LVA or T-type) and HVA currents (Fig. 2A, top panel), whereas another subset of cells presented HVA currents only (Fig. 2A, bottom panel). The mean cell capacitance between these 2 populations was not significantly different (LVA + HVA: 27.5 ± 3.8 pF vs HVA: 24.2 ± 2.1 pF; P = 0.4352). In cells exhibiting both LVA and HVA conductances, T-type currents were enhanced after chronic exposure to homocysteine. For instance, the mean peak T-type current density was increased by 2.9-fold (P = 0.0392) in homocysteine-treated cells (−35.5 ± 9.5 pA/pF, n = 14) as compared to control cells (−12.4 ± 2.1 pA/pF, n = 12) (Fig. 2B). By contrast, we observed a nonsignificant trend (P = 0.1308) toward larger peak HVA current densities in cells treated with homocysteine (from −15.8 ± 3.2 pA/pF, n = 12 to −25.5 ± 4.5 pA/pF, n = 14) (Fig. 2B). The mean peak membrane potential remained unaltered for both LVA (−50.4 ± 1.3 vs −50.0 ± 1.5 mV; P > 0.9999) and HVA currents (−17.3 ± 1.2 vs −19.6 ± 2.1 mV; P = 0.4854) (Fig. 2C). In cells displaying HVA currents only, chronic exposure to homocysteine had no significant effect on the calcium conductance (P = 0.5697) and peak current potential (P = 0.8530) (Figs. 2D and E). Furthermore, the relative proportion of cells displaying both LVA and HVA currents was not altered (chi-squared test, P = 0.7651) in homocysteine condition (23% vs 27%), suggesting that homocysteine did not trigger expression of LVA currents in cells that did not initially express T-type channels (Fig. 2F). In contrast to the effect of homocysteine on VGCCs, we did not observe significant alterations of voltage-gated sodium channels. For instance, the mean maximum slope sodium conductance in homocysteine-treated cells (1252 ± 265 pS/pF, n = 6) was comparable (P = 0.9048) to the one in control cells (1329 ± 177 pS/pF, n = 6) (data not shown).

Figure 2.

Figure 2.

Altogether, these data indicate that homocysteine enhances LVA T-type calcium currents in cultured nociceptors.

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3.3. Pharmacological inhibition of T-type channels reverses mechanical allodynia associated with homocysteinemia

To assess whether T-type channels contribute to mechanical allodynia associated with homocysteinemia, we examined mechanical withdrawal threshold in homocysteinemic rats after intraplantar injection of the selective T-type channel blocker TTA-A2. Although intraplantar injection of TTA-A2 did not affect mechanical withdrawal threshold in control animals (TTA-A2: 11.1 ± 2.4 g/mm2 vs DMSO: 10.1 ± 2.2 g/mm2 at 20 minutes, n = 7; P = 0.9499), it significantly increased mechanical withdrawal threshold in homocysteinemic animals (TTA-A2: 10.9 ± 1.7 g/mm2 vs DMSO: 5.5 ± 0.6 g/mm2 at 20 minutes, n = 7; P = 0.0076) to a level that was not different (P = 0.8019) from control animals (Fig. 3A). Similar results were observed with the T-type channel blockers ML218 (Fig. 3B) and mibefradil (Fig. 3C).

Figure 3.

Figure 3.

Altogether, these data indicate that T-type channel activity is required for homocysteinemia-induced mechanical allodynia.

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3.4. Homocysteine enhances surface expression of v3.2">Cav3.2 channels

Next, we performed a series of experiments to analyze the molecular mechanism by which homocysteine enhances T-type currents. It is well established that the T-type current in DRG neurons is largely carried by v3.2">Cav3.2 channels,9 although the presence of Cav3.3 has also been documented.16,66 Therefore, we first determined whether the effect of homocysteine observed in nociceptive neurons can be recapitulated in tsA-201 cells expressing v3.2">Cav3.2 channels. Consistent with the data obtained in DRG neurons, we observed that chronic exposure to homocysteine enhanced T-type currents in tsA-201 cells expressing v3.2">Cav3.2 channels (Fig. 4A). For instance, in response to a depolarizing pulse to −20 mV, a 1.6-fold increase (P < 0.0001) in the mean peak T-type current density was observed in cells cultured with homocysteine (−41.3 ± 2.7 pA/pF, n = 81) compared to control cells (−25.5 ± 2.0 pA/pF, n = 83) (Fig. 4B). The voltage dependence of v3.2">Cav3.2 channels underwent only a minor although significant shift of ∼3 mV (P = 0.0004) toward more negative potentials (−48.7 ± 0.7 mV, n = 81) compared to control cells (−45.5 ± 0.7 mV, n = 83) (Fig. 4B, inset). The mean maximum slope conductance was increased by 57% (P < 0.0001) in cells treated with homocysteine (905 ± 59 pS/pF, n = 81) vs control cells (575 ± 42 pS/pF, n = 83) (Fig. 4C). We also observed a dose-dependent effect of homocysteine, where a significant potentiation of the T-type current by 19% (P = 0.0372) was observed after exposure to 30-μM homocysteine, and reached a maximum at 100 μM (Fig. 4C). By contrast, homocysteine had no effect on Cav3.1, Cav3.3, and Cav2.2 channels (Fig. S1, available online as supplemental digital content at http://links.lww.com/PAIN/A855).

Figure 4.

Figure 4.

We next determined whether homocysteine acts from the outside or from the inside of the cell. Cellular uptake of homocysteine occurs through system A and glutamate transporters that are inhibited by 2-MEA and L-glutamic acid (L-Glu), respectively.31 Although application of MEA and L-Glu had no significant effect on v3.2">Cav3.2 channels in control cells, it fully prevented the enhancement of the T-type current in cells treated with homocysteine, demonstrating that homocysteine-dependent potentiation of the T-type current occurs from the inside of the cell (Fig. 4C). Because the enhancement of T-type currents could have also resulted from a homocysteine-dependent redox modulation of v3.2">Cav3.2,46 we challenged the redox-insensitive v3.2">Cav3.2 H191Q mutant with chronic exposure to homocysteine. Under these experimental conditions, homocysteine-dependent potentiation still persisted such that the mean maximal macroscopic T-type conductance was increased by 78% (P = 0.0024) (1206 ± 141 pS/pF, n = 20) compared to control cells (675 ± 98 pS/pF, n = 20) (Fig. 4D). Furthermore, acute external application of homocysteine had no effect on the T-type current (Fig. 4E), ruling out the occurrence of a redox modulation. Altogether, these data indicate that the phenotypical effect of homocysteine observed on native T-type currents in DRG neurons is recapitulated in tsA-201 cells expressing v3.2">Cav3.2 channels, and support the validity of this experimental system to further investigate the mechanism by which homocysteine modulates T-type currents.

Next, we determine whether the enhancement of T-type currents occurred as a result of an increase in the density of v3.2">Cav3.2 in the plasma membrane, or as a consequence of a gain of function in the gating properties of the channel. Nonstationary noise analysis of the T-type current revealed a substantial increase in the number of functional channels in cells cultured with homocysteine (Fig. 4F). For instance, the number of functional channels (N) was increased by 74% (P = 0.0095) in homocysteine-treated cells (736 ± 87, n = 24) compared to control cells (421 ± 58, n = 22) (Fig. 4G, left panel). By contrast, the single channel conductance (γ) and the channel opening probability (Po) remained unaltered (Fig. 4G, middle and right panels, respectively). The increased channel density in the plasma membrane was further confirmed by surface immunostaining using the exofacial hemagglutinin (HA)-tagged v3.2">Cav3.2. The expression of HA-v3.2">Cav3.2 quantified from low-magnification images obtained from nonpermeabilized cells revealed a 43% increase (P = 0.0079) in cells treated with homocysteine as compared to control cells (Figs. 4H and I). By contrast, immunostaining performed on permeabilized cells (Figs. 4H and I) as well as immunoblot from total cell lysates (Figs. 4J and K) showed no difference in the total expression of the channel.

Altogether, these data support the notion that homocysteine augments T-type currents by enhancing the density of v3.2">Cav3.2 channels in the plasma membrane.

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3.5. Homocysteine potentiates the recycling of v3.2">Cav3.2 back to the plasma membrane

The steady-state expression of v3.2">Cav3.2 at the cell surface is the net result between the number of channels reaching to and being removed from the plasma membrane. To further examine the detailed mechanisms by which homocysteine enhances surface expression of v3.2">Cav3.2, we first assessed the transport of the channels through intracellular organelles by fluorescence recovery after photobleaching (FRAP) at physiological temperature (37°C) in live tsA-201 cells expressing mCherry-tagged v3.2">Cav3.2. Cells were cotransfected with either an endoplasmic reticulum (ER)-targeted green fluorescent protein (ER-GFP) (Fig. S2A, available online as supplemental digital content at http://links.lww.com/PAIN/A855) or a Golgi-targeted GFP (Golgi-GFP) (Fig. S2E, available online as supplemental digital content at http://links.lww.com/PAIN/A855). Our FRAP measurements revealed a slight increase in the subcellular distribution of the channels in homocysteine-treated cells (Fig. S2B and F, available online as supplemental digital content at http://links.lww.com/PAIN/A855). For instance, the normalized mobile fraction in the ER was increased by 18% (P = 0.0256) in cells treated with homocysteine (78 ± 5%, n = 20) as compared to control cells (60 ± 4%, n = 16) (Fig. S2C, available online as supplemental digital content at http://links.lww.com/PAIN/A855). A similar increase of 19% (P = 0.0013) was also observed in the Golgi apparatus (76 ± 4%, n = 25) as compared to control cells (57 ± 3%, n = 20) (Fig. S2G, available online as supplemental digital content at http://links.lww.com/PAIN/A855). However, the FRAP kinetics remained unchanged (Fig. S2D and H, available online as supplemental digital content at http://links.lww.com/PAIN/A855) suggesting that the intracellular dynamics of v3.2">Cav3.2 may not be responsible for the increased surface density of the channel. In addition, the kinetics of channel internalization from the plasma membrane at 37°C in homocysteine-treated cells (τ = 79 ± 5 minutes, n = 3) were comparable (P = 0.4000) to those measured in control cells (τ = 97 ± 8 minutes, n = 3) (Fig. S3, available online as supplemental digital content at http://links.lww.com/PAIN/A855). Therefore, we next assessed the potential contribution of the endosomal pathway using a dominant negative mutant of Rab11 (Rab11S25N) to disrupt the recycling of proteins from the endosome to the plasma membrane as previously documented.3 In control cells, overexpression of Rab11S25N caused a 40% decrease (P = 0.0286) in the density of v3.2">Cav3.2 in the plasma membrane demonstrating that a significant fraction of channels is indeed recycled back to the cell surface (Figs. 5A and B). Remarkably, overexpression of Rab11S25N completely precluded the effect of homocysteine such that surface expression of v3.2">Cav3.2 in Rab11S25N-expressing cells and treated with homocysteine was similar (P > 0.9999) to nontreated cells (Figs. 5A and B). Consistent with this observation, overexpression of Rab11S25N also precluded homocysteine-induced increase in T-type currents (Fig. 5C). For instance, the mean maximum slope conductance in homocysteine-treated Rab11S25N-expressing cells (468 ± 57 pS/pF, n = 18) was similar (P = 0.8025) to nontreated cells (448 ± 56 pS/pF, n = 17) (Fig. 5C).

Figure 5.

Figure 5.

Altogether, these results support a mechanism by which homocysteine enhances surface expression of v3.2">Cav3.2 channels by potentiating endosomal recycling of the channel back to the plasma membrane.

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3.6. Homocysteine enhances v3.2">Cav3.2 channels through a protein kinase C–dependent signaling pathway

Previous studies in several cell types have reported that homocysteine triggers PKC activation.4,62,79 Consistent with these findings, we observed that PKC activity is increased by 2.2-fold (P < 0.0001) in tsA-201 cells treated with homocysteine compared to control cells (Figs. 6A and B). We next assessed the level of phosphorylation of v3.2">Cav3.2 channels in homocysteine-treated cells. Immunoprecipitation experiments using an antiphosphoserine antibody revealed an increase in phosphorylation of v3.2">Cav3.2 in cells treated with homocysteine (Figs. 6C and D). For instance, the phosphorylation level of v3.2">Cav3.2 was increased by 3.3-fold (P = 0.0286) in cells treated with homocysteine as compared to control cells (Fig. 6D). Therefore, we investigated the possibility that PKC may participate in the potentiation of v3.2">Cav3.2 by homocysteine. We reasoned that if activation of PKC underlies homocysteine-induced increase in v3.2">Cav3.2 surface expression, pharmacological preactivation of PKC should prevent or minimize the subsequent effect of homocysteine. Indeed, in the presence of phorbol 12-myristate 13-acetate (PMA), a specific PKC activator, homocysteine-dependent potentiation of the T-type current was abolished and the mean maximum slope conductance of cells expressing v3.2">Cav3.2 channels and treated with homocysteine was similar to vehicle-treated cells (P = 0.5424) (Fig. 6E). Because PMA also triggers the production of reactive oxygen species (ROS)68 that could have interfered with the regulation of v3.2">Cav3.2, we examined the effect of homocysteine in cells treated with 50-μM hydrogen peroxide (H2O2) that mimics the production level of ROS induced by PMA (Fig. S4A, available online as supplemental digital content at http://links.lww.com/PAIN/A855). In cells treated with H2O2, homocysteine still enhanced T-type currents supporting the notion that PMA abolished the effect of homocysteine through preactivating PKC (Fig. S4B, available online as supplemental digital content at http://links.lww.com/PAIN/A855). Data obtained with PMA were further confirmed using the constitutively active mutant PKCmuS738/742E (PKCCA). Similar to what was observed with PMA, homocysteine-induced increase in T-type currents was abolished in cells expressing PKCCA, confirming that the effect of homocysteine relies on the activation of PKC (Fig. 6F).

Figure 6.

Figure 6.

Altogether, these data indicate that homocysteine-induced increase in T-type currents requires the activation of PKC and is associated with an increase phosphorylation of v3.2">Cav3.2 channels.

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3.7. Identification of v3.2">Cav3.2 phosphorylation sites responsible for homocysteine-induced increase in T-type currents

To identify PKC-dependent phosphorylation loci within the v3.2">Cav3.2 channel responsible for the effect of homocysteine, we first used the NetPhos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/) to predict the potential PKC sites within the v3.2">Cav3.2 amino acid sequence as previously described.8 The predicted phosphorylation sites were then selected based on these previously reported to be effectively phosphorylated both in HEK293T cells and in brain cells.7 By combining these 2 theoretical and empirical approaches, we consequently identified 4 PKC-dependent phosphorylation sites at serines S532, S653, S1144, and S2188 within the v3.2">Cav3.2 channel (Fig. 7A). We then disrupted these PKC phosphorylation sites by replacing serine (S) with alanine (A) and assessed the conductance of phosphorylation-deficient v3.2">Cav3.2 channels exposed to homocysteine (Figs. 7B–F). Although homocysteine still enhanced the maximal conductance of cells expressing the S653A mutant channel by 72% (137 ± 14 pS/pF, n = 31, vs 236 ± 34 pS/pF, n = 29, P = 0.0071) (Figs. 7D and G), application of homocysteine on cells expressing the S532A, S1144A, and S2188A mutant channels had no significant effect on the T-type current (Figs. 7C and E–G). A similar effect was observed when glycosylation-deficient v3.2">Cav3.2 mutants were coexpressed with the constitutively active PKC mutant (PKCCA) (Fig. S5A-F, available online as supplemental digital content at http://links.lww.com/PAIN/A855). Consistent with this observation, disruption of PKC-dependent phosphorylation loci at serine S532, S1144, and S2188 also prevented homocysteine-dependent potentiation of v3.2">Cav3.2 surface expression (Figs. 7H and I). By contrast, surface expression of the S653A mutant remained enhanced in the presence of homocysteine (Figs. 7H and I). The observation that surface expression of the S653A mutant in the presence of homocysteine was higher than expected from the T-type current may suggest that this mutation might render the channel susceptible to a reduction in channel function in the presence of homocysteine, and that the S653 residue may play a more complex modulatory role of the overall homocysteine effects. It is also worth noting that small alterations in the expression level and/or activation properties of the phosphorylation-deficient mutants were also observed; however, these aspects were not further investigated in this study (Fig. S6, available online as supplemental digital content at http://links.lww.com/PAIN/A855).

Figure 7.

Figure 7.

Altogether, these results are consistent with the notion that several PKC-dependent phosphorylation sites within the v3.2">Cav3.2 are required for homocysteine-dependent potentiation of the channel.

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4. Discussion

Aberrant upregulation of v3.2">Cav3.2 channels in primary afferent fibers is linked to a number of chronic pain conditions resulting from various types of traumatic nerve injury,33,75,78 toxic neuropathies induced by chemotherapy agents,23,48 as well as painful diabetic neuropathy.14,34,35,44 The mechanisms by which v3.2">Cav3.2 channels are upregulated under pathological conditions is subject to ongoing research, and a number of mechanisms have already been reported.17,26,36,37,49,53,59,70,73 In this study, we show that T-type currents in tsA-201 cells expressing v3.2">Cav3.2 channels are enhanced in the presence of elevated levels of homocysteine. Previous studies have reported that v3.2">Cav3.2 channels are sensitive to redox modulation both in vivo and in vitro, a regulation that relies on histidine 191 located in the outer IS3-IS4 loop of the channel and, in principle, this could have accounted for the phenotypical effect of homocysteine observed in our experiments.46,67 However, 3 lines of evidence support the notion that redox-dependent modulation of v3.2">Cav3.2 did not play a significant role in this effect. First, acute external application of homocysteine did not produce significant alteration of channel activity. This finding seems to contrast with previous work from Pathirathna et al.51 who reported redox effects of homocysteine on v3.2">Cav3.2 channels. However, these authors examined the effects of homocysteine on native T-type currents in DRG neurons, whereas we tested acute effects on heterologously expressed human v3.2">Cav3.2 channels. Second, homocysteine-dependent potentiation of v3.2">Cav3.2 channels was entirely abolished in the presence of system A and glutamate transporter inhibitors, the 2 main pathways by which homocysteine enters the cells,31 indicating that homocysteine-dependent potentiation of v3.2">Cav3.2 relies on an intracellular signaling pathway. Third, the redox-insensitive v3.2">Cav3.2 H191Q mutant retained the ability to undergo homocysteine-dependent potentiation. By contrast, we have shown that enhanced T-type currents in the presence of homocysteine results from an increased channel density in the plasma membrane. In addition, we show that homocysteine enhances surface expression of v3.2">Cav3.2 by potentiating the recycling of the channel back to the cell surface, without affecting the total pool of v3.2">Cav3.2. Indeed, our data revealed that homocysteine-induced increase in v3.2">Cav3.2 expression is prevented by a dominant negative mutant of Rab11, a recognized experimental strategy to evaluate the recycling of plasma membrane proteins from late endosomes.3

It is well established that protein phosphorylation affects diverse aspects of ion channel function.40 Here, and consistent with previous reports,4,62,79 we show that basal PKC activity is enhanced in the presence of homocysteine, parallel with increased phosphorylation of v3.2">Cav3.2. Our observation that homocysteine did not produce an additional effect when applied on cells pretreated with the PKC activator PMA or in cells expressing a constitutive active mutant of PKC revealed that homocysteine-induced upregulation of v3.2">Cav3.2 relies on PKC activity rather than an additional signaling pathway that would have produced a cumulative effect. In addition, our detailed analysis of PKC-dependent phosphorylation of v3.2">Cav3.2 identified 3 potential phosphorylation loci at serines S532, S1144, and S2188, located within the I-II loop, II-III loop, and carboxy-terminal region of the channel, respectively, and essential for homocysteine to mediate upregulation of v3.2">Cav3.2. Consequently, disrupting any of these loci was sufficient to abolish homocysteine-induced upregulation of v3.2">Cav3.2. This notion was further supported by the observation that coexpression of a constitutively active mutant of PKC enhanced the expression of v3.2">Cav3.2 WT, but not the expression of phosphorylation-deficient channel mutants. Although these data are consistent with the notion that enhanced expression of v3.2">Cav3.2 in the presence of homocysteine relies on the direct phosphorylation of the channel by PKC, quantitative phosphoproteomic analysis of v3.2">Cav3.2 will be required to further confirm that indeed phosphorylation at these specific loci is enhanced by homocysteine. In addition, several studies have reported either a stimulatory C on v3.2">Cav3.2 expression.50,55,61,80,81 One possible explanation is the involvement of diverse PKC isoforms that may either phosphorylate v3.2">Cav3.2 at different loci or phosphorylate channel modulators, which may account for different phenotypical effects. Consistent with this notion, the phosphorylation loci identified in this study differ from those previously identified to modulate the gating properties of v3.2">Cav3.2,7 indicating that phosphorylation at specific loci controls distinct aspects of v3.2">Cav3.2 function. It remains to be determined how these loci act together in concert within v3.2">Cav3.2 to support the potentiation of the channel by homocysteine. One possibility is that phosphorylation of v3.2">Cav3.2 could produce a dynamic rearrangement of intramolecular interactions within the channel protein, which in turn may modulate the ability of the channel to engage into the recycling pathway.

In addition to our in vitro data, we have shown that homocysteine is causally linked to the development of PN. For instance, we show that mechanical withdrawal threshold in a rat model of prenatal homocysteinemia is decreased, indicative of the development of mechanical allodynia. It has been previously reported that plantar injection of reducing cysteine analogs including L-homocysteine induces thermal hyperalgesia in rats, which partially relies on T-type channel activity.51 Here, we did not observe any alteration of thermal nociception in our animal model of prenatal homocysteinemia. However, the 2 models reflect strikingly different conditions. Although local injection of homocysteine produces transient alterations of peripheral nociceptors, our animal model of chronic systemic homocysteinemia, which to some extent mimics the human metabolic condition, reflects long-term physiological alterations, which are likely to have resulted from a more permanent cellular remodeling.

Two lines of evidence suggest that v3.2">Cav3.2 channels may play a role in the development of mechanical allodynia in our animal model of homocysteinemia. First, similar to what we observed with recombinant v3.2">Cav3.2 channels, T-type currents in cultured DRG neurons was enhanced in the presence of homocysteine. By contrast, we did not observe any alteration of the voltage-gated sodium conductance that could have otherwise contributed to an overall increase in nociceptors excitability, and likely would have altered thermal nociception. Second, mechanical allodynia was reversed on pharmacological inhibition of T-type channels by intraplantar injection of various T-type channel blockers including TTA-A2, ML218, and mibefradil. These data are consistent with previous reports documenting the implication of peripheral v3.2">Cav3.2 channels in mechanotransduction.24 The observation that pharmacological inhibition of T-type channels did not alter basal mechanical threshold suggests that the contribution of T-type channels in mechanonociception is more prominent in homocysteinemic conditions, possibly because of an increased channel expression. However, we cannot rule out the possibility that T-type channels may simply contribute to a pathological signaling pathway involving downstream effectors.

To conclude, T-type channels are implicated in several neuronal conditions,74 and clinical studies have suggested that homocysteinemia could represent a risk factor for PN. However, this notion has never been investigated experimentally. Here, we present evidence that homocysteinemia is causally linked to the development of mechanical allodynia, and that T-type channels may play an important role in this condition. Nevertheless, it remains to be seen whether enhanced T-type currents in DRG neurons relies on the same PKC-dependent signaling pathway we described in vitro. In addition, it is likely that homocysteine may have additional cellular effects,12,13 which could have contributed to the increased mechanical nociception in homocysteinemic animals. Regardless of the exact underlying mechanisms, our observation that homocysteinemia is causally linked to the development of PN, and potentially to other neurological conditions, may have important clinical implications. Indeed, dietary supplementation with vitamin B-complex and folic acid to normalize homocysteine levels may potentially be effective while being devoid of adverse effects, therefore presenting an incomparable benefit/risk ratio.

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Disclosures

The authors report no conflicts of interest.

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Supplemental video content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/A855.

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Supplemental video content

Video content associated with this article can be found online at http://links.lww.com/PAIN/A856.

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Acknowledgements

The authors thank Drs Stephen Ferguson (university of Ottawa) and Angelika Hausser (University of Stuttgart) for providing cDNAs encoding for Rab11S25N and PKCmuS738/742E, respectively. The authors thank Dr Guzel Ziyatdinova (Kazan federal University) for her help with the measurement of homocysteine level.

N. Weiss is supported by the Institute of Organic Chemistry and Biochemistry (IOCB). A. Tomin is supported by an IOCB postdoctoral fellowship. G.F. Sitdikova and E.V. Gerasimova are supported by the Russian Science Foundation. G.W. Zamponi is a Canada Research Chair and supported by the Canadian Institutes of Health Research.

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Keywords:

Homocysteinemia; Homocysteine; Pain; Allodynia; Calcium channel; T-type channel; v3.2">Cav3.2

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