Objective: To construct phosphorylation sites domain (PSD) mutant of myristoylated alanine-rich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) SAC.Methods: Human pkcental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. SerlS9, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM3 and PSD mutant on the synthesis and secretion of MUCSAC induced by cold, respectively. Results: Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0-MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUCSAC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUCSAC than that of control group (P< 0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05).Conclusion: TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.%目的:构建豆蔻酰化富丙氨酸激酶c底物(MARCKS)磷酸化位点(PSD)突变体,并探讨瞬时受体电位M8离子通道(TRPM8)及MARCKS在冷刺激诱导黏蛋白(MUC)5AC合成及胞吐过程中发挥的作用.方法:利用PCR克隆全编码MARCKS目的基因,并通过定点突变技术将PSD的4个丝氨酸突变为天冬氨酸编码基因.将野生型及突变体cDNA亚克隆至pcDNA3.0载体后用酶切及DNA测序鉴定.以TRPM8特异性阻断剂BCTC、重组突变质粒转染人气道上皮16HBE细胞为干预手段,用免疫荧光及ELISA检测二者对冷空气刺激诱导的16HBE细胞合成及分泌MUC5AC的影响.结果:成功构建了pcDNA3.0-MARCKS重组质粒及pcDNA3.0-MARCKS-PSD突变体.冷刺激组胞内合成及分泌MUC5AC水平显著高于未刺激对照组(P＜0.05).与冷刺激组比较,BCTC可显著抑制由冷刺激诱导的MUC5AC合成及分泌(P＜0.05)；与冷刺激组比较,转染MARCKS-PSD突变cDNA可显著下调由冷刺激诱导的MUC5AC高分泌水平(P＜0.05),同时胞内的MUC5AC水平与对照组比较呈现显著升高状态(P＜0.01),而转染载体及野生型MARCKS对冷刺激诱导的MUC5AC胞内合成及胞外分泌水平无显著影响(p＞0.05).结论:TRPM8及MARCKSPSD的磷酸化过程介导了冷刺激诱导气道上皮细胞内MUC5AC的胞吐过程.
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