| Objective: Calcium channels are composed of several transmembrane proteins. They control the process of calcium influx into intracellular strictly, and can produce electri-chemical signals which are necessary for physiology of cells.Calcium Channel Blocker (CCB) has a selective inhibition effect on calcium influx into intracellular via calcium channel. Thus CCB can release vascular smooth muscle, decrease peripheral vascular resistance, and in the end depress the blood pressure. Meantime, CCB possess a negative inotropic effect that inhibits contractility and conduction of cardiac myocyte.There are various classes of CCB, mainly play their effect on L-type calcium channels. The drug binding sites are on theα1c subunit according to which CCB is divided into three subclasses: Dihydropyridines, Benzothiazepines and Phenylalkylamines.Dihydropyridines (DHPs) have been applied widely, for example, in respects of vasodilation, bronchiectasis, anti-arteriosclerosis, anti-diabetes, anti-gene mutagenesis and anti-convulsion etc.Although DHPs were primarily developed as cardiovascular agents, several have been used for other medical applications. For example, nimodipine is used as an anti-ischemic agent in the treatment of Alzheimer's disease and other dementias, migraine and cerebral hemorrhagic vasospasm. Besides, Nifedipine is used in the treatment of migraine, hypertrophic cardiomyopathy and Raynaud's phenomenon; it could also be used in the treatment of diabetic neuropathy.It is meaningful that make sure to utilize maximum curative effect of CCB meanwhile to lower its side effects. Therefore, inner allosteric of calcium channel and mechanism of CCB became research priorities. Generally, LTCC has three states: open (O), inactivation (I) and close (C) state. Inactivation contains voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Dynamics analysis showed that DHPs inhibit LTCC mainly through binding with VDI. Detailed mechanisms have been not known yet.Timothy Syndrome arises from a sporadic single nucleotide change that generates a mutation (G436R) in the pore-forming subunit of the L-type Ca2+ channel—CaV1.2. And VDI of TS-LTCC is significantly slowed compared with WT-LTCC.To make a detailed mechanism of VDI, we analyzed the characteristics of VDI and investigated a particular mechanism of CCB by comparing TS-LTCC with WT-LTCC.Methods:1 Preparation of channel proteins:α2δ,β2 subunit and a point mutation ofα1c subunit——G436R2 HEK293 cells were incubated in DMEM containing 10% fetal bovine serum at 37℃in 5% CO2/95% air in vitro. A stable cell line ofα2δandβ2 subunit was constructed by antibiotics screening. WT-LTCC or TS-LTCC was transfected with GFP into the stable cell line.3 Pipettes were pulled into a macro electrode for use. Intracellular fluid and different extracellular fluid were made as showed below. The protocol of patch clamp experiment was programmed as showed below. All the data were recorded in a whole-cell mode.4 VDI, recovery of LTCC and a dose-response curve of Nifedipine were analyzed respectively by IGOR6.0 software.5 All the statistical treatments were taken using SPSS 11.0 software.Results:We established a stable cell line ofα2δandβ2 subunit by antibiotics screening. This stable cell line had decreased error of data in a large content. Characteristics of inactivation were analyzed as follows: WT-LTCC: tf = 496.23ms, ts = 3016.11ms, R1 = 0.29, R20 =0.004, Relative Af = 0.71, Relative As = 0.29 and Relative A0 = 0.01. TS-LTCC: tf = 1270.51 ms, ts = 6358.5 ms, R1 = 0.67, R20 =0.08, Relative Af = 0.29, Relative As = 0.65 and Relative A0 = 0.05. Characteristics of recovery were analyzed as follows: WT-LTCC: tf = 1.60s, ts = 13.40s, Relative Af = 0.21, Relative As = 0.74. TS-LTCC: no tf, ts = 25.90s, no Relative Af, Relative As = 0.94. By using different concentrations of Nifedipine, dose-response curve of WT-LTCC and TS-LTCC were measured. Surprisingly, two similar values of IC50 were acquired, about 1nmol/ml. This result exposed that Nifedipine inhibits LTCC mainly by stabilizing the slow inactivation of the channel.Conclusion:1 Compared with WT-LTCC which had two inactivation parts—the fastone and the slow one, TS-LTCC only had a slow inactivation, the fast part totally vanished.2 Sensitivity to Nifedipine showed no significant differences between WT-LTCC and TS-LTCC.3 Nifedipine inhibits LTCC mainly by stabilizing the slow inactivation of the channel.
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