| T-type calcium channels, also known as the low voltage-activated calcium channels(LVA), are expressed in various tissues and play key roles in physiology and pathology such as neuronal firing, pain, and cancer. Hence, it is important to understand the mechanisms for T-type calcium channels regulation.However, the research on T-type calcium channels regulation is limited due to the lack of specific modulators. While substantial literature suggests many hormones or neurotransmitters can modulate T-type calcium channels via activation of G protein-coupled receptors(GPCRs), the underlying molecular mechanisms remain unclear.Here in this research we focused our study on the modulation of T-type calcium channels in dorsal root(DRG) neurons by G protein-coupled receptors(GPCRs). In addition, we also studied the effect of hydrogen sulfide(H2S) on T-type calcium currents. Part oneGPCRs modulation of T-type calcium channels in DRG neurons and theunderlying mechanisms.Objective: To study the modulation of T-type calcium channels by activation of GPCRs and the underlying molecular mechanisms.(1) To observe the effect of the μ opioid receptor agonist DAMGO on T-type calcium currents recorded from rat DRG neurons.(2) To observe the effect of the GABAB receptor agonist baclofen on T-type currents recorded from rat DRG neurons and the CaV3.2 currents recorded in HEK293 cells.(3) To explore the molecular mechanisms for modulation of T-type calcium channels by baclofen.Methods: Whole-cell patch clamp technique was used.Results:(1) The μ opioid agonist DAMGO(250 n M) reversibly inhibited T-type(LVA) and high voltage-activated calcium channel(HVA) in rat DRG neurons by 41.8 ± 10.4%(n = 3) and 65.8 ± 6.7%(n = 8), respectively.(2) HVA calcium currents in rat DRG neurons were inhibited by DAMGO in a concentration-dependent manner, with an EC50 of 17.72 ± 6.9 n M.(3) DAMGO did not inhibit T-type calcium currents in some DRG neurons.(4) The GABAB receptor against baclofen(10 μM) reversibly inhibited T-type(LVA) and HVA calcium currents in DRG neurons by 22.53 ± 2.05%(n = 15) and 31.90 ± 3.11%(n = 27), respectively. The inhibition could be partly blocked by 400 ng/ml PTX(P < 0.001, n = 9~11).(5) The inhibition of baclofen on HVA calcium currents could be partly blocked by 2 m M GDP-beta-s(P < 0.05, n = 16), but GDP-beta-s had no effect on the inhibition of baclofen on LVA calcium currents(P > 0.05, n = 6).(6) The inhibition of baclofen on LVA calcium currents could be partly recovered by the reducing agent DTT(1 m M)(P < 0.01, n = 6), while DTT had no effect on the inhibition of baclofen on HVA calcium currents(P > 0.05, n = 14).(7) Baclofen(10 μM) reversibly inhibited CaV3.2 calcium currents heterologously expressed in HEK293 cells, co-expressed with GABAB receptor, by 24.73 ± 1.11%(n = 12). The inhibition could be partly recovered by the reducing agent DTT(1 m M)(P < 0.001, n = 7). Conclusion: Using whole cell patch clamp, we demonstrated that T-type calcium currents can be inhibited by DAMGO, an agonist of μ opioid receptor, and baclofen, the agonist of GABAB receptors. However, we found that most of the rat DRG neurons which expressed functional T-type calcium channels did not express μ opioid receptor. GABAB receptor-induced modulation of T-type calcium currents were most likely mediated by Gαi/o protein, with also possible involvement of redox pathway. Part twoThe effect of hydrogen sulfide(H2S) on T-type calcium currents in DRGneuronsObjective: To observe the effect of H2 S on the T-type calcium currents in rat DRG neuronsMethods: Whole-cell patch clamp technique was used.Results:(1) The H2 S donor, Na HS at concentration of 100μM, 300μM, 1 m M inhibited T-type calcium currents in DRG neurons concentration-dependently. Na HS had no effect on HVA calcium currents(P > 0.05, n = 11).(2) Na HS at higher concentrations of 3 m M and 10 m M activated rather than inhibited T-type calcium current in rat DRG neurons.(3) Further experiments demonstrated the augment of T-type calcium currents at high concentration by Na HS was induced by Na+ and was not related to H2 S itself. When 10 m M Na HS was changed to 10 m M Na Cl, the latter also increased T-type calcium currents. However, the effect of 10 m M Na HS seemed larger than that of 10 m M Na Cl(P = 0.07, n = 5).Conclusion: H2 S can inhibit T-type calcium currents in rat DRG neurons. High concentration of Na+ in Na HS can activate T-type calcium currents. |
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