The Effect Of Glycosylation On Ca_v3.2 T-type Calcium Chanel

Objective: Glycosylation is one of post-translational modifications of proteins,and play an important role in maintaining the normal structure and regulating the function.Recently,we research the effect of N-type glcosylation,induced by tunicamycin,on Ca_v3.2 T-type Calcium channel by whole-cell patch clamp.Methods:1 The cultivate of the cells.The cell line used in the study is HEK 293 transfecting with human Ca_v3.2-T.2 Whole-cell patch clamp technique.The Ca_v3.2-T current was recorded by whole-cell patch clamp at room temperature.The protocal of activiation curve: the holding potential was-100 mV,the test potential was from-100 mV to +50mV with a 5 mV step voltage.The duration of test pulse was 300 ms,the sweep interval was 2s.the protocal of inactivation cure: The 500 ms prepulse was from-100 mV to-20 mV,the test potential was-20 mV.Results: The tunicamycin has a strongly inhibitory of temporal correlation on Ca_v3.2-T current.In 6 hours with tunicamycin,the Ca_v3.2-T current doesn't change.With prolong to 12 hours,the current has reduce.And more reduction appeared after 24 hours with tunicamycin.The current density of control group and tunicamycin group with tunicamycin for 1 hour was40.98± 3.98pA/pF(n=14)and-40.70±3.26pA/pF(n=12,P=0.957).The current density of control group and tunicamycin group with tunicamycin for 6hour was-41.22±3.78pA/pF(n=18)and-41.81±3.96 p A/p F(n=15,P=0.916).The current density of control group and tunicamycin group with tunicamycin for 12 hour was 41.00±4.26pA/pF(n=11)and 18.54±2.68pA/pF(n=9,P <0.001).The current density of control group and tunicamycin group withtunicamycin for 24 hour was-44.32±1.91pA/pF(n=12)and13.06±1.59pA/pF(n=17,P<0.001).Meanwhile,the steady activation curves of Ca_v3.2-T channel shifted in a hyperpolarized direction after 12 hours with tunicamycin.The half activated potential of control group and tunicamycin group with tunicamycin for 1 hour was-44.24±1.28mV(n=14)and-44.83±2.01mV(n=12,P=0.799).The half activated potential of control group and tunicamycin group with tunicamycin for 6 hour was-44.15±0.386mV(n=18)?-43.70±0.493mV(n=15,P=0.470).The half activated potential of control group and tunicamycin group with tunicamycin for 12 hour was 44.76±0.99mV(n=11)and 42.17±0.53mV(n=9,P=0.045).The half activated potential of control group and tunicamycin group with tunicamycin for 24 hour was 44.13±1.03mV(n=12)and 39.09±0.65mV(n=17,P<0.001).Conclusions: Tunicamycin inhibits Ca_v3.2-T ion channel current by blocking N-linked glycosylation.In the meantime,Tunicamycin also shifted the steady activation curve,but no effect the steady inactivation curve.So that,the window current of Ca_v3.2-T channel significantly reducted.These changes will significantly affect the physiological and pathological functions of T-type calcium channel.