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Mol Pain
2018 Jan 01;14:1744806918765806. doi: 10.1177/1744806918765806.
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Peripheral nerve injury increases contribution of L-type calcium channels to synaptic transmission in spinal lamina II: Role of α2δ-1 subunits.
Alles SR
,
Garcia E
,
Balasubramanyan S
,
Jones K
,
Tyson JR
,
Joy T
,
Snutch TP
,
Smith PA
.
Abstract
Background Following peripheral nerve chronic constriction injury, the accumulation of the α2δ-1 auxiliary subunit of voltage-gated Ca2+ channels in primary afferent terminals contributes to the onset of neuropathic pain. Overexpression of α2δ-1 in Xenopus oocytes increases the opening properties of Cav1.2 L-type channels and allows Ca2+ influx at physiological membrane potentials. We therefore posited that L-type channels play a role in neurotransmitter release in the superficial dorsal horn in the chronic constriction injury model of neuropathic pain. Results Whole-cell recording from lamina II neurons from rats, subject to sciatic chronic constriction injury, showed that the L-type Ca2+ channel blocker, nitrendipine (2 µM) reduced the frequency of spontaneous excitatory postsynaptic currents. Nitrendipine had little or no effect on spontaneous excitatory postsynaptic current frequency in neurons from sham-operated animals. To determine whether α2δ-1 is involved in upregulating function of Cav1.2 L-type channels, we tested the effect of the α2δ-1 ligand, gabapentin (100 µM) on currents recorded from HEK293F cells expressing Cav1.2/β4/α2δ-1 channels and found a significant decrease in peak amplitude with no effect on control Cav1.2/β4/α2δ-3 expressing cells. In PC-12 cells, gabapentin also significantly reduced the endogenous dihydropyridine-sensitive calcium current. In lamina II, gabapentin reduced spontaneous excitatory postsynaptic current frequency in neurons from animals subject to chronic constriction injury but not in those from sham-operated animals. Intraperitoneal injection of 5 mg/kg nitrendipine increased paw withdrawal threshold in animals subject to chronic constriction injury. Conclusion We suggest that L-type channels show an increased contribution to synaptic transmission in lamina II dorsal horn following peripheral nerve injury. The effect of gabapentin on Cav1.2 via α2δ-1 may contribute to its anti-allodynic action.
Figure 1. Effects of nitrendipine on sEPSC and eEPSCs recorded from lamina II neurons of CCI and sham-operated rats. Cumulative probability plots for effect of nitrendipine on inter-event interval (IEI) of sEPSCs from sham-operated animals (a) and those subject to CCI (b). (c and d) Same data replotted as mean IEI. Cumulative probability plots for effect of nitrendipine on amplitude of sEPSCs from sham-operated animals (e) and those subject to CCI (f). (g and h) same data replotted as mean IEI. For shams, IEI data for 1629 events and nitrendipine data for 1398 events in 24 neurons. For CCI, IEI data for 1853 events and nitrendipine data for 1379 events in 27 neurons. For shams, amplitude data for 1561 events and nitrendipine data for 1415 events in 24 neurons. For CCI, amplitude data for 1898 events and nitrendipine data for 1389 events in 27 neurons. Note that p values were derived from K-S test for cumulative probability graphs and paired t-test for the bar graphs. **p < 0.001 and *p < 0.05 compared to pre-drug amplitude (paired t-test). In each case, recordings were obtained from only one neuron per slice. Sample recordings of eEPSCs following DRZ stimulation from sham-operated animals (i) and those subject to CCI (j), before, during, and after superfusion of 2 µM nitrendipine. (k) Time course of effect of nitendipine on eEPSC amplitude in 13 neurons from sham-operated animals and 12 neurons form animals subject to CCI. Error bars = SEM. **p < 0.001 and *p < 0.05 compared to pre-drug amplitude (paired t-test). CCI: chronic constriction injury; eEPSC: evoked excitatory postsynaptic currents.
Figure 2. Effects of nitrendipine on PWT in animals subject to CCI. (a) Averaged data from five animals, arrows labeled N indicate times of IP injections of 5 mg/kg nitrendipine. Dashed line with gray symbols illustrates minimal effect of vehicle injections (two animals) (b) Data from each animal at the 36 h time point to illustrate the variation in nitrendipine effectiveness. PWT: paw withdrawal threshold.
Figure 3. Gabapentin block of L-type Cav1.2 channel currents expressed in HEK293F cells. (a) Time course of inhibitory effect of 100 µM GBP on Ba2+ currents recorded from HEK cells expressing Cav1.2 channels in combination with either α2δ–1 (i) or α2δ–3 (ii) auxiliary subunits. The averaged fraction of peak current amplitude is plotted against time. Cells were perfused with GBP during the periods indicated by horizontal bars. Inserts correspond to representative macroscopic currents obtained before (black) and after (red) the application of GBP. (iii) The fraction of inhibition after 3 min of drug application is shown as a bar graph. Number of cells examined are indicated in brackets; the asterisk indicates a statistical significance p< 0.05. (b) Normalized current-voltage curves (i) obtained for the recombinant Cav1.2 channel in the presence (red symbols) and in the absence (black symbols) of 100 µM GBP. The values of fitted parameters corresponding to the continuous lines obtained by Boltzmann function fits are shown. Application of GBP produced an average current inhibition of ∼25% with no significant changes in the voltage dependence parameters. Data are plotted as mean ± SEM (n = 5 for each data point). (ii) Typical traces from a representative cell show the effect of GBP on the I-V relationship with no apparent modification of current kinetics.
Figure 4. Gabapentin inhibits a dihydropyridine-sensitive inward current in undifferentiated PC12 cells. a. Current-voltage relation of barium currents were examined in the presence of 1 mM ω-Conotoxin GVIA and 200nM ω-Agatoxin VIA to eliminate the contribution of native non-L-type channels. Currents were elicited by voltage ramps from −100 to +100mV at 1mV/ms, and cells were exposed to 100nM of the agonist (S)-(-)-BayK 8644 prior to the application of 100 µM GBP. b. The bar graph shows the average of peak amplitude values (n = 5) obtained in cells perfused with external recording solution (Ba2+ 20mM), compared to that obtained after the consecutive addition of peptide toxins, BayK 8644 and GBP. Data are plotted as Mean ± S.E.M. The asterisk indicates the change in current amplitude after perfusion with GBP is statistically significant at p < 0.05. c. Endogenous expression of ancillary α2δ subunits (Left) and HVA α1 subunits (Right) in PC12 cells was quantified by RT-PCR. Expression levels were calculated relative to the “housekeeping gene” GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). Mean values ± S.D. of three samples.
Figure 5. Effects of GBP on sEPSCs recorded from lamina II neurons of CCI and sham-operated rats. Cumulative probability plots for effect of 100 µM GBP on (a) inter-event interval (IEI) of sEPSCs in sham-operated animal and (b) in animals subject to CCI. (c and d) same data replotted as bar graphs. Cumulative probability plots for effect of 100 µM GBP on (e) amplitude of sEPSCs in sham-operated animal and (f) in animals subject to CCI. (g and h) same data replotted as bar graphs. For sham-operated rats, control data for 515 events and GBP data for 625 events in 7 neurons from 5 rats. For CCI ,control data for 886 events and GBP data for 721 events in 9 neurons from 8 rats. p-values were derived from K-S test for cumulative probability graphs and paired t-test for the bar graphs. **p < 0.001 and *p < 0.05 compared to pre-drug amplitude (paired t-test). In each case, recordings were obtained from only one neuron per slice. sEPSC: spontaneous excitatory postsynaptic currents; CCI: chronic constriction injury.
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