Effects Of Capsazepine On The Human Hyperpolarization-activated Cyclic Nucleotide-gated 2 (hHCN2) Channel And The HHCN4 Channel

Objective: Capsazepine can inhibit the neuropathic pain-related hyperpolarization- activated cyclic nucleotide-gated 1 (HCN1) channel, and therefore may be clinically applied for treating neuropathic pain. However, the possible effects of capsazepine on HCN2 and HCN4 channels, which are mainly expressed in the heart and involved in the control of cardiac rhythmicity, have rarely been reported. The present study aimed at exploring the effects of capsazepine on HCN2 and HCN4 channels.Methods: (1) Construction of eukaryotic expressing vector of human HCN2 and HCN4 gene; (2) Restriction enzyme digestion analysis and the DNA sequence analysis; (3) pIRES2-HCN2-EGFP,pIRES2-HCN4-EGFP plasmid and the empty plasmid (pIRES2-EGFP) was transfected into HEK293 cells by Lipofacta mine 2000, respectively; (4) Green fluorescent protein GFP expression was determined by inverted fluorescence microscopy ; (5) HCN2 mRNA and HCN4 mRNA expression in HEK293 cells were determined by reverse transcription-polymerase chain reaction assay (reverse transcriptase PCR, RT-PCR) . Designing specific primers, PCR amplification of corresponding fragments; (6) G418 selection to obtain stable expression cell line hHCN2 and hHCN4; (7) HCN2 and HCN4 channel currents were determined with the whole-cell patch-clamp technique, perfusion with different concentrations of capsazepine, observed current changes.Results: (1) pIRES2-HCN2-EGFP and pIRES2-HCN4-EGFP plasmid were successfully constructed, DNA sequence analysis confirmed that the HCN2 and HCN4 gene was completely accurate, Restriction enzyme digestion analysis confirmed that DNA fragments consistent with the size of the target gene sequence; (2) The green Fluorescence expression was observed in transfection of pIRES2-HCN2-EGFP, pIRES2-HCN4-EGFP plasmid and empty plasmid (pIRES2- EGFP) HEK293 cells with inverted fluorescence microscope, while non-transfected HEK293 cells did not see the green fluorescence; (3) The size of 230bp and 233bp fragments were amplified in transfected pIRES2-HCN2-EGFP, pIRES2-HCN4- EGFP plasmid HEK293 cells, control group showed no corresponding specific fragment; (4) After 600μg/μl G418 screening 3-4 weeks, stable expression cell line hHCN2 and hHCN4 were obtained; (5) The inward hyperpolarization currents, which were readily blocked by 2 mM Cscl and 40μM ZD7288, were recorded from HEK293 cells expressing HCN2 and HCN4 channels, confirming that HCN2/4 acted as If channels in HEK293 cells, control group was detected no hyperpolarization inward current; (6) Capsazepine blocked HCN2 and HCN4 Channel currents: (i) capsazepine block HCN2 and HCN4 currents, IC50 (Half- maximal inhibition) valμes were 6.1μM and 5.8μM, between the 0.1μM-10μM, the inhibition rate with the concentrations increased, while more than 50μM, the inhibition rate of increase in concentration did not change significantly; (ii) At -130mv , HCN2 and HCN4 channel current densities were -29.46±3.0 pA/pF (n = 10) and -31.51±3.44 pA/pF (n = 8), respectively; After perfusion with capsazepine , the HCN2 and HCN4 current densities were -19.67±4.6 pA/pF (n = 8, P < 0.05) and -17.86±4.1 pA/pF (n = 6, P<0.05), respectively; (iii) the activation curve of HCN2/4 channel becomes more hyperpolarization with capsazepine.Conclusions: (1) EGFP as a reporter gene can be detected fast tracking protein; (2) pIRES2-HCN2-EGFP, pIRES2-HCN4-EGFP plasmid were transfected into HEK293 cells. After G418 resistant screening, stable cell line expressing hHCN2 and hHCN4 were obtained, establishing heterologous expression of HCN2 and HCN4 channel model. Using the model, we can be easily conduct further studies to properties of HCN channels and develop drug target for HCN channels; (3) Capsazepine inhibits HCN2 and HCN4 channel currents, clinical application of it for treatment the neurogenic pain may affect HCN2 and HCN4 associated with side effects; (4) Capsazepine inhibits HCN2 and HCN4 channel current in a concentration dependent manner, and makes two-channel activation curve toward the hyperpolarizing direction, its mechanism may be related with the channel protein transmembrane segment (S2-S4) .