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Original Research

Reversal of Neuropathic Pain in Diabetes by Targeting Glycosylation of Cav3.2 T-Type Calcium Channels

  1. Peihan Orestes1,3,
  2. Hari Prasad Osuru1,
  3. William E. McIntire4,
  4. Megan O. Jacus1,
  5. Reza Salajegheh1,
  6. Miljen M. Jagodic1,
  7. WonJoo Choe1,6,
  8. JeongHan Lee1,7,
  9. Sang-Soo Lee8,9,
  10. Kirstin E. Rose1,
  11. Nathan Poiro1,
  12. Michael R. DiGruccio1,3,
  13. Katiresan Krishnan5,
  14. Douglas F. Covey5,
  15. Jung-Ha Lee8,9,
  16. Paula Q. Barrett4,
  17. Vesna Jevtovic-Todorovic1,2,3 and
  18. Slobodan M. Todorovic1,2,3⇑
  1. 1Department of Anesthesiology, University of Virginia Health System, Charlottesville, Virginia
  2. 2Department of Neuroscience, University of Virginia Health System, Charlottesville, Virginia
  3. 3Neuroscience Graduate Program, University of Virginia Health System, Charlottesville, Virginia
  4. 4Department of Pharmacology, University of Virginia Health System, Charlottesville, Virginia
  5. 5Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri
  6. 6Department of Anesthesiology and Pain Medicine, InJe University, Ilsan Paik Hospital & College of Medicine, Goyang-City, South Korea
  7. 7Department of Anesthesiology and Pain Medicine, Busan Paik Hospital, InJe University, College of Medicine, Busan, South Korea
  8. 8Department of Life Science, Sogang University, Seoul, South Korea
  9. 9Interdisciplinary Program of Biotechnology, Sogang University, Seoul, South Korea
  1. Corresponding author: Slobodan M. Todorovic, st9d{at}virginia.edu.
Diabetes 2013 Nov; 62(11): 3828-3838. https://doi.org/10.2337/db13-0813
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  • FIG. 1.
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    FIG. 1.

    NEU and PNG modulate recombinant human CaV3.2 channels. A: Traces represent families of T-currents evoked in representative HEK-293 cells in control conditions (top panel) and after incubation of 1.5 units/mL NEU at 37°C for 3 h (lower panel) by voltage steps from –90 mV (Vh) to Vt from −80 through −20 mV in 5-mV increments. Bars indicate calibration. B: Average normalized I-V curves (current/maximum current, I/Imax) are shown in HEK-293 cells in control conditions (n = 18) and after incubations of NEU (n = 13). C and D: We measured time-dependent activation (10–90% rise time [C]) and inactivation τ (single exponential fit of decaying portion of the current waveforms [D]) from I-V curves in HEK-293 cells (B) over the range of test potentials from −50 mV to 10 mV. There are differences in up to twofold slower times between the control and NEU groups at each tested potential. *Significance of P < 0.05. E: Bars represent 10–90% current activation rise times and current inactivation τ (Vh = −90 mV, Vt = −30 mV) measured in control cells (n = 13) and cells incubated with PNG (20 units/mL at 37°C for 12 h) (n = 7). Note that PNG-treated cells had slower activation and inactivation kinetics. *Significance of P < 0.01. F: Bar graphs depict peak current density (Vh = −90 mV, Vt = −30 mV) measured in multiple HEK-293 cells in control conditions (n = 20) and cells after incubation of NEU alone (n = 18), PNG alone (n = 7), and combined PNG and NEU (n = 11). Note that all three treatments significantly decreased peak current density compared with control cells: control 95 ± 10 (open bar, n = 20), NEU 65 ± 8 (n = 18, P < 0.01), PNG 59 ± 2 (n = 7, P < 0.01), and NEU plus PNG 53 ± 5 (n = 11, P < 0.01). *Significance of P < 0.05. Vertical bars in all panels represent SEM from multiple determinations.

  • FIG. 2.
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    FIG. 2.

    Molecular mechanisms of glycosylation of CaV3.2 channels. A: Schematic diagram of Cav3.2 showing the position of conserved putative N-glycosylation sites in the extracellular face of the channel in domains I and III. Designated asparagine residues (in red bold fonts) were mutated to alanine residues. B: Representative traces in gray show nickel inhibition of T-current (black traces) in HEK-293 cell transiently transfected with WT CaV3.2 (top) and N192Q CaV3.2 (bottom) channels. In both experiments, 100 μmol/L NiCl2 was applied in the bath. On average, nickel blocked 97 ± 2% of inward currents of N192Q CaV3.2 (n = 5) and WT CaV3.2 (n = 6) channels. Bars indicate calibration. C: Bar graph represents the average effect of N192Q CaV3.2 mutation compared with WT CaV3.2 T-current kinetics (Vh = −90 mV, Vt = −30 mV) in HEK-293 cells. On average, N192Q mutant has slower 10–90% rise times by ~60% (8.2 ± 0.8 s) compared with WT CaV3.2 currents (5.1 ± 0.2 s). Similarly, on average N192Q mutant has slower inactivation τ-values by ~50% (31.4 ± 2.2 s) compared with WT CaV3.2 currents (21.8 ± 0.6 s). Vertical lines are ±SEM of multiple determinations. Number of cells in each experiment is indicated in parentheses. *Significance of P < 0.001.

  • FIG. 3.
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    FIG. 3.

    Altered membrane expression of putative glycosylation sites in CaV3.2 channels. A: Representative confocal images in the left panel show HEK-293 cells transiently transfected with WT CaV3.2 channels where concanavalin A immunofluorescence is represented in red color. Middle panel shows green immunofluorescence representing EGFP-tagged CaV3.2 transfection in the same cells. Right panel shows merged images where overlay is presented in yellow color. B: Representative confocal images in the left panel show HEK-293 cells transiently transfected with N192Q CaV3.2 channels where concanavalin A immunofluorescence is represented in red color. Middle panel shows green immunofluorescence representing EGFP-tagged N192Q CaV3.2 transfection in the same cells. Right panel shows merged images where overlay is presented in yellow color. C: Representative confocal images in the left panel show HEK-293 cells transiently transfected with N271Q CaV3.2 channels where concanavalin A immunofluorescence was represented in red color. Middle panel shows green immunofluorescence representing EGFP-tagged N271Q CaV3.2 transfection in the same cells. Right panel shows merged images where overlay is presented in yellow color. D: Representative confocal images in the left panel show HEK-293 cells transiently transfected with N1466Q CaV3.2 channels where concanavalin A immunofluorescence was represented in red color. Middle panel shows green immunofluorescence representing EGFP-tagged N1466Q CaV3.2 transfection in the same cells. Right panel shows merged images where overlay is presented in yellow color. Note that EGFP and concanavalin A show very little overlap in their subcellular distributions. E: Bar graphs represent average values from multiple experiments (those depicted in A–D). Colocalization values for EGFP and concanavalin epifluorescence were quantified and compared between mutants and WT CaV3.2 channels. Only N1466Q mutants (dark-gray column, 9.9 ± 3.7, n = 4) displayed ~73% decrease in colocalization value compared with WT channels (black column, 36.2 ± 3.8, n = 4). In contrast, colocalization values of N192Q (open column, n = 4) and N271Q mutants (light gray bar, n = 3) were not significantly different from WT CaV3.2. **Statistically significant difference from CaV3.2 WT channels (P < 0.01). NS (not significant), P > 0.05 compared with CaV3.2 WT channels. Calibration bars are marked on all panels.

  • FIG. 4.
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    FIG. 4.

    Biochemical evidence of glycosylation of CaV3.2 channels. Immunoblotting with FLAG antibody reveals a shift in apparent molecular weight of NH2-terminal fragment of CaV3.2 channel (bottom arrow) but not full channel (top arrow). Note that treatment with PNGase-F and Endo F1 but not Endo F2 and Endo F3 caused an obvious change in mobility of FLAG-labeled NH2-terminal fragment of CaV3.2 from ~60 to 50 KDa. Using mass spectrometry, we confirmed that the deglycosylated NH2-terminal fragment of CaV3.2 channels recognized by FLAG antibodies contains most of the repeat I of CaV3.2 channel (data not shown). N-term., NH2-terminal.

  • FIG. 5.
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    FIG. 5.

    Alterations of macroscopic T-current kinetics in acutely dissociated small DRG cells from diabetic ob/ob mice. The data show original T-current traces (Vh −90 mV, Vt −80 mV through −30 mV) from representative DRG cells from a healthy WT mouse (A) and a diabetic ob/ob mouse (B). The averaged data show marked acceleration in T-current inactivation (C) and activation (D) kinetics in ob/ob mice compared with age-matched WT mice. Data are averages of multiple cells (WT n = 27, ob/ob n = 20) ±SEM. *P < 0.05. Solid lines on C and D are single exponential fits to experimental data points. E: Normalized peak T-current activation curves from similar experiments shown in A and B. Number of cells is indicated in the parentheses. Solid black lines are fitted using equation 1, giving half-maximal activation (V50), which occurred at −46.6 ± 0.6 mV with a k of 6.3 ± 0.5 mV in WT mice. Similarly, V50 was –47.6 ± 0.7 mV with a k of 7.4 ± 0.6 mV in the DRG cells from ob/ob mice. F: Normalized peak T-current steady-state inactivation curves. T-currents are evoked by test steps to −30 mV after 3.5-s prepulses to potentials ranging from −110 mV to −45 mV in 5-mV increments. Number of cells is indicated in the parentheses. Solid black lines are fitted using equation 2, giving V50, which occurred at −75.0 ± 0.4 mV with a k of 8.9 ± 0.4 mV in WT mice. Similarly V50 was –75.8 ± 0.4 mV with a k of 8.2 ± 0.3 mV in the DRG cells from ob/ob mice. The voltage dependencies of activation and steady-state inactivation were described with single Boltzmann distributions of the following forms where Imax is the maximal activatable current, V50 is the voltage where half the current is activated or inactivated, and k is the voltage dependence (slope) of the distribution. Embedded Image (1) Embedded Image (2), where exp = ex.

  • FIG. 6.
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    FIG. 6.

    NEU treatment in vitro reversed kinetic alterations and normalized T-current density in small DRG cells from diabetic ob/ob mice. A: Traces represent families of T-currents evoked in representative DRG cells in a WT mouse (top panel) and a diabetic ob/ob mouse after incubation of 1.5 units/mL of NEU at 37°C for 3 h (lower panel) by voltage steps from Vh = −90 mV to Vt from −80 through −25 mV in 5-mV increments. Bars indicate calibration. Bar graphs with the averaged data show that NEU treatments completely reversed DRG T-current density (Vh = −90 mV, Vt = −30 mV) (B), T-current inactivation measured by inactivation τ (C), and activation kinetics measured by 10–90% rise time (D) in ob/ob mice compared with healthy WT mice. Control was compared with post-NEU treatments. DRG cells were freshly dissociated as noted in Fig. 5. Recordings were performed at room temperature while NEU was incubated for 1–3 h at 37°C. Control cells were treated with saline. Data are averages of multiple cells (as indicated in parenthesis) ±SEM. *P < 0.001; n.s., not significant, P > 0.05.

  • FIG. 7.
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    FIG. 7.

    NEU treatment in vivo reversed mechanical hyperalgesia in diabetic ob/ob mice. A: The graph shows average data points indicating that i.pl. injections of NEU but not saline into right paws completely reversed mechanical hyperalgesia in diabetic ob/ob mice. Arrow indicates time point of i.pl. injections. *Significant change of PWRs with P < 0.05 compared with baseline PWR prior to i.pl. injections. B: The graph shows average data points indicating that i.pl. injections of NEU and saline into right paws did not affect PWRs in the left paws of diabetic ob/ob mice. Arrow indicates time point of i.pl. injections. C: The graph shows average data points indicating that i.pl. injections of NEU but not saline into right paws caused transient hyperalgesia in healthy WT mice at the time point of 30 min. Arrow indicates time point of i.pl. injections. *Significant change of PWRs with P < 0.001 compared with baseline PWR prior to i.pl. injections. D: The graph shows average data points indicating that i.pl. injections of NEU and saline into right paws did not affect PWRs in the left paws of WT mice. Arrow indicates time point of i.pl. injections. E: The graph with averaged data from eight experiments shows that intraperitoneal injections (thin arrow) of selective T-channel blocker ECN (25 mg/kg i.p.) completely reversed mechanical hyperalgesia in diabetic ob/ob mice as evidenced by decreased PWRs in both right and left paws (#P < 0.001). Subsequent i.pl. injections (thick arrow) of 1.5 units/mL NEU in the same animals did not significantly influence new baseline values of mechanical PWRs at a time point of 90 min, but it significantly increased PWRs at time points of 120, 150, and 180 min (*P < 0.05). F: The graph with averaged data from eight experiments shows that intraperitoneal injections (thin arrow) of vehicle used to dissolve ECN (Cyc) did not affect mechanical hyperalgesia in diabetic ob/ob mice as evidenced by stable PWRs in both right and left paws at a time point of 60 min. Subsequent i.pl. injections (thick arrow) of 1.5 units/mL NEU in the same animals effectively reversed diabetic hyperalgesia by significantly decreasing PWRs at time points of 120, 150, and 180 min (*P < 0.001).

  • FIG. 8.
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    FIG. 8.

    NEU treatment in vivo reversed thermal hyperalgesia in diabetic ob/ob mice. A: The bar graphs with averaged data show that i.pl. injections of NEU completely reversed thermal hyperalgesia in diabetic ob/ob mice, while the same treatment was ineffective in WT mice. NEU was injected into right (R) paws, while uninjected left (L) paws served as controls. PWLs were determined in mice before (time point 0) and 10 and 30 min after injections of 10 μL NEU (arrows) into hind paws. Data are averages of seven experiments ±SEM. Symbols indicating significance of NEU treatments are as follows: *P < 0.01 for right vs. left paws at the same time points, and †P < 0.01 for data points at 30 min after NEU injections vs. 0 min. B: The bar graphs with averaged data show that injections of selective T-channel blocker ECN at 25 mg/kg i.p. completely reversed thermal hyperalgesia in diabetic ob/ob mice, as evidenced by elevated PWLs in both right and left paws (P < 0.001). Subsequent intraplantar injections of 1.5 units/mL NEU in the same animals did not significantly alter new baseline values of thermal PWLs (n.s., P > 0.05, n = 4). Arrows indicate times of injections of ECN and NEU.

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Reversal of Neuropathic Pain in Diabetes by Targeting Glycosylation of Cav3.2 T-Type Calcium Channels
Peihan Orestes, Hari Prasad Osuru, William E. McIntire, Megan O. Jacus, Reza Salajegheh, Miljen M. Jagodic, WonJoo Choe, JeongHan Lee, Sang-Soo Lee, Kirstin E. Rose, Nathan Poiro, Michael R. DiGruccio, Katiresan Krishnan, Douglas F. Covey, Jung-Ha Lee, Paula Q. Barrett, Vesna Jevtovic-Todorovic, Slobodan M. Todorovic
Diabetes Nov 2013, 62 (11) 3828-3838; DOI: 10.2337/db13-0813

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Reversal of Neuropathic Pain in Diabetes by Targeting Glycosylation of Cav3.2 T-Type Calcium Channels
Peihan Orestes, Hari Prasad Osuru, William E. McIntire, Megan O. Jacus, Reza Salajegheh, Miljen M. Jagodic, WonJoo Choe, JeongHan Lee, Sang-Soo Lee, Kirstin E. Rose, Nathan Poiro, Michael R. DiGruccio, Katiresan Krishnan, Douglas F. Covey, Jung-Ha Lee, Paula Q. Barrett, Vesna Jevtovic-Todorovic, Slobodan M. Todorovic
Diabetes Nov 2013, 62 (11) 3828-3838; DOI: 10.2337/db13-0813
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