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Effects of Capsazepine on the Human Hyperpolarization-activated Cyclic Nucleotide-gated 2 (hHCN2) Channel and the hHCN4 Channel

Author: ZuoGuangFeng
Tutor: ChenShaoLiang
School: Nanjing Medical University
Course: Internal Medicine
Keywords: Pepper Ping (Capsazepine) Human HCN2 and HCN4 channels Eukaryotic expression vector
CLC: R285
Type: Master's thesis
Year: 2011
Downloads: 30
Quote: 1
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Abstract


Objective: capsaicin receptor blocker pepper flat (Capsazepine) involved in neuropathic pain HCN1 channels have blocking effect, may be used for the clinical treatment of neuropathic pain, but the chili level whether the expression of the heart and the major participate in the formation of cardiac rhythm channel HCN2 and HCN4 impact has not yet been reported. This study aimed to investigate the pepper flat on HCN2 and HCN4 channel effects. Methods: (1) HCN2 and HCN4 construct eukaryotic expression vector; (2) DNA sequencing and restriction maps verification; (3) liposome respectively pIRES2-HCN2-EGFP, pIRES2-HCN4-EGFP plasmid and load plasmid (pIRES2-EGFP) were transfected into HEK293 cells; (4) inverted fluorescence microscope expression of green fluorescent protein (GFP); (5) reverse transcription - polymerase chain reaction (reverse transcriptase PCR, RT-PCR) detection of HEK293 cells HCN2 mRNA and HCN4 mRNA expression, and specific primers, PCR amplification of the corresponding fragment; (6) G418 selection HCN2 and HCN4 stably expressing cell lines; (7) Whole-cell patch-clamp technique detection HCN2 and HCN4 channel current, perfused with different concentrations of pepper flat, observe current changes. Results: (1) successfully constructed pIRES2-HCN2-EGFP and pIRES2-HCN4-EGFP plasmid, DNA sequencing confirmed that HCN2 and HCN4 gene sequence is normal, no mutation nucleotides, DNA fragments with restriction map shows the size of the target gene sequence consistent; ( 2) inverted fluorescence microscope, observed transfected pIRES2-HCN2-EGFP, pIRES2-HCN4-EGFP plasmid and empty plasmid transfection (pIRES2-EGFP) in HEK293 cells were GFP expression plasmid untransfected HEK293 cells not seen green fluorescence; (3) transfected pIRES2-HCN2-EGFP, pIRES2-HCN4-EGFP plasmid transfected HEK293 cells were amplified 230bp and 233bp fragments load group and the control group specific fragment HEK293 cells showing no corresponding ; (4) 600μg/μl G418 screening 3-4 weeks, HCN2 and HCN4 stably expressing cell lines; (5) the experimental group HEK293 cells were detected on 2 mM CsCl and 40μM ZD7288 sensitive inward hyperpolarizing current , confirmed in HEK293 cells HCN2 / 4 plays a role If channel, no-load group and the control group not detected current; (6) pepper flat block HCN2 and HCN4 channel current: (i) flat block HCN2 and pepper HCN4 channel currents IC50 (half-maximal inhibition) values ??were 6.1μM and 5.8μM, 0.1μM-10μM in between, the inhibition rate increased with increasing concentration, more than 50μM, inhibition rate increased concentration values ??did not change significantly; ( ii) to -130 mV when, HCN2 and HCN4 channel current density was -29.46 ± 3.0 pA / pF (n = 10) and -31.51 ± 3.44 pA / pF (n = 8), after perfusion pepper flat, HCN2 and HCN4 channel current density was -19.67 ± 4.6 pA / pF (n = 8, P lt; 0.05) and -17.86 ± 4.1p A / pF (n = 6, P lt; 0.05); (iii) chili level so that the HCN2 / 4-channel activation curve in the hyperpolarizing direction. Conclusions: (1) EGFP as a reporter gene can be detected fast tracking protein; (2) pIRES2-HCN2-EGFP, pIRES2-HCN4-EGFP plasmid was transfected into HEK293 cells screened by G418 resistance obtained stable expression hHCN2 and hHCN4 cell lines established heterologous expression HCN2 and HCN4 channels model, based on this model can be easily carried out on ion channels as well as to carry out more in-depth study to HCN channels as therapeutic targets for drug development; (3 ) Pepper Ping inhibition of human HCN2 and HCN4 channel current clinical application HCN1 channels are involved in the treatment of neuropathic pain may cause HCN2 and HCN4 function associated with side effects; (4) pepper flat inhibit channel currents were HCN2 and HCN4 concentration-dependent, and makes two kinds of channel activation curve in the hyperpolarizing direction, and its mechanism may be associated with the channel protein transmembrane segments (S2-S4) related.

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